Therapeutic monoclonal antibodies that neutralize botulinum neurotoxins

ABSTRACT

This invention provides antibodies that specifically bind to and neutralize botulinum neurotoxin type A (BoNT/A) and the epitopes bound by those antibodies. The antibodies and derivatives thereof and/or other antibodies that specifically bind to the neutralizing epitopes provided herein can be used to neutralize botulinum neurotoxin and are therefore also useful in the treatment of botulism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. Ser. No.60/648,256, filed Jan. 27, 2005, which is incorporated herein byreference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support by Grant No: AI53389 andAI56493, awarded by the National Institutes of Health, and by Departmentof Defense Grants DAMD17-03-C-0076 and DAMD17-98-C-8030. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

This invention relates antibodies that neutralize botulinum neurotoxins(e.g., BoNT/A) and their use in the treatment of botulism.

BACKGROUND OF THE INVENTION

Botulism is caused by botulinum neurotoxin secreted by members of thegenus Clostridium and is characterized by flaccid paralysis, which ifnot immediately fatal requires prolonged hospitalization in an intensivecare unit and mechanical ventilation. Naturally occurring botulism isfound in infants or adults whose gastrointestinal tracts becomecolonized by Clostridial bacteria (infant or intestinal botulism), afteringestion of contaminated food products (food botulism), or in anaerobicwound infections (wound botulism) (Center for Disease Control (1998)Botulism in the United States, 1899-1998. Handbook for epidemiologists,clinicians, and laboratory workers. Atlanta, Ga. U.S. Department ofHealth and Human Services, Public Health Service: downloadable at“bt.cdc.gov/agent/botulism/index.as.asp”. Botulism neurotoxins (BoNTs)are also classified by the Centers for Disease Control (CDC) as one ofthe six highest-risk threat agents for bioterrorism (the “Category Aagents”), due to their extreme potency and lethality, ease of productionand transport, and need for prolonged intensive care (Arnon et al.(2001) JAMA 285: 1059-1070). Both Iraq and the former Soviet Unionproduced BoNT for use as weapons (United Nations Security Council (1995)Tenth report of the executive committee of the special commissionestablished by the secretary-general pursuant to paragraph 9(b)(J) ofsecurity council resolution 687 (1991), and paragraph 3 of resolution699 (1991) on the activities of the Special Commision; Bozheyeva et al.(1999) Former soviet biological weapons facilities in Kazakhstan: past,present, and future. Center for Nonproliferation Studies, MontereyInstitute of International Studies), and the Japanese cult Aum Shinrikyoattempted to use BoNT for bioterrorism (Arnon et al. (2001) supra). As aresult of these threats, specific pharmaceutical agents are needed forprevention and treatment of intoxication.

No specific small molecule drugs exist for prevention or treatment ofbotulism, but an investigational pentavalent toxoid vaccine is availablefrom the CDC (Siegel (1988) J. Clin. Microbiol. 26: 2351-2356) and arecombinant vaccine is under development (Smith (1998) Toxicon 36:1539-1548). Regardless, mass civilian or military vaccination isunlikely due to the rarity of disease or exposure and the fact thatvaccination would prevent subsequent medicinal use of BoNT.Post-exposure vaccination is useless, due to the rapid onset of disease.Toxin neutralizing antibody (Ab) can be used for pre- or post-exposureprophylaxis or for treatment (Franz et al. (1993) Pp. 473-476 In B. R.DasGupta (ed.), Botulinum and Tetanus Neurotoxins: Neurotransmission andBiomedical Aspects. Plenum Press, New York). Small quantities of bothequine antitoxin and human botulinum immune globulin exist and arecurrently used to treat adult (Black and Gunn. (1980) Am. J. Med., 69:567-570; Hibbs et al. (1996) Clin. Infect. Dis., 23: 337-340) and infantbotulism (Arnon (1993). Clinical trial of human botulism immuneglobulin, p. 477-482. In B. R. DasGupta (ed.), Botulinum and TetanusNeurotoxins: Neurotransmission and Biomedical Aspects. Plenum Press, NewYork) respectively.

Recombinant monoclonal antibody (mAb) could provide an unlimited supplyof antitoxin free of infectious disease risk and not requiring humandonors for plasmapheresis. Given the extreme lethality of the BoNTs,mAbs must be of high potency in order to provide an adequate number ofdoses at reasonable cost. The development of such mAbs has become a highpriority research aim of the National Institute of Allergy andInfectious Diseases. While to date no single highly potent mAbs havebeen described, we recently reported that combining two to three mAbscould yield highly potent BoNT neutralization (Nowakowski et al. (2002)Proc. Natl. Acad. Sci. USA, 99: 11346-50).

The development of mAb therapy for botulism is complicated by the factthat there are at least seven BoNT serotypes (A-G) (Hatheway (1995)Curr. Top. Microbio. Immunol, 195: 55-75.) that show little, if any,antibody cross-reactivity. While only four of the BoNT serotypesroutinely cause human disease (A, B, E, and F), there has been onereported case of infant botulism caused by BoNT C (Oguma et al. (1990)Lancet 336: 1449-1450), one outbreak of foodborne botulism linked toBoNT D (Demarchi, et al. (1958) Bull. Acad. Nat. Med., 142: 580-582),and several cases of suspicious deaths where BoNT G was isolated(Sonnabend et al. (1981) J. Infect. Dis., 143: 22-27). AerosolizedBoNT/C, D, and G have also been shown to produce botulism in primates bythe inhalation route (Middlebrook and Franz (1997) Botulinum Toxins,chapter 33. In F. R. Sidell, E. T. Takafuji, D. R. Franz (eds.), MedicalAspects of Chemical and Biological Warfare. TMM publications,Washington, D.C.), and would most likely also affect humans. Thus it islikely that any one of the seven BoNT serotypes can be used as abiothreat agent.

Variability of the BoNT gene and protein sequence within serotypes hasalso been reported and there is evidence that such variability canaffect the binding of monoclonal antibodies to BoNT/A (Kozaki et al.(1998) Infect. Immun., 66: 4811-4816; Kozaki et al. (1995) Microbiol.Immunol., 39: 767-774). It is currently not clear the extent of suchtoxin variability within the different serotypes, nor its impact on thebinding and neutralization capacity of monoclonal antibody panels.

SUMMARY OF THE INVENTION

This invention pertains to antibodies that bind to and neutralizebotulinum neurotoxin(s). We have discovered that particularly effectiveneutralization of a Botulism neurotoxin (BoNT) serotype can be achievedby the use of neutralizing antibodies that bind two or more subtypes ofthe particular neurotoxin serotype with high affinity. While this can beaccomplished by using two or more different antibodies directed againsteach of the subtypes, in certain embodiments even more efficientneutralization is achieved by the use of one or more antibodies whereeach antibody is cross-reactive with at least two BoNT subtypes. Incertain embodiments this invention provides for compositions comprisingneutralizing antibodies that bind two or more BoNT subtypes (e.g.,BoNT/A1, BoNT/A2, BoNT/A3, etc.) with high affinity.

Thus, in one embodiment, this invention provides a method ofneutralizing botulinum neurotoxin in a mammal (e.g., a human). Themethod typically involves administering to the mammal at least twodifferent neutralizing antibodies for a BoNT serotype, wherein at leastone of the two antibodies binds at least two different subtypes of saidBoNT serotype (e.g., BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F,etc.) with an affinity greater than about 10 nM. In certain embodimentsat least one of the antibodies binds at least two different subtypesselected from the group consisting of BoNT/A1, BoNT/A2, BoNT/A3, andBoNT/A4, each with an affinity greater than about 10 nM. In certainembodiments at least one of the antibodies binds BoNT/A1 and BoNT/A2each with an affinity greater than about 10 nM. In certain embodimentsboth antibodies simultaneously bind at least one, preferably at leasttwo of the subtypes. In certain embodiments the antibodies each compriseat least one, at least two, at least three, at least 4, at least five,or at least six CDRs selected from the group consisting of RAZ1 VL CDR1,RAZ1 VL CDR2, RAZ1 VL CDR3, RAZ1 VH CDR1, RAZ1 VH CDR2, RAZ1 VH CDR3,1D11 VL CDR1, 1D11 VL CDR2, 1D11 VL CDR3, 1D11 VH CDR1, 1D11 VH CDR2,1D11 VH CDR3, 2G11 VL CDR1, 2G11 VL CDR2, 2G11 VL CDR3, 2G11 VH CDR1,2G11 VH CDR2, 2G11 VH CDR3, 5G4 VL CDR1, 5G4 VL CDR2, 5G4 VL CDR3, 5G4VH CDR1, 5G4 VH CDR2, 5G4 VH CDR3, 3D12 VL CDR1, 3D12 VL CDR2, 3D12 VLCDR3, 3D12 VH CDR1, 3D12 VH CDR2, 3D12 VH CDR3, CR1 VL CDR1, CR1 VLCDR2, CR1 VL CDR3, CR1 VH CDR1, CR1 VH CDR2, CR1 VH CDR3, CR2 VL CDR1,CR2 VL CDR2, CR2 VL CDR3, CR2 VH CDR1, CR2 VH CDR2, CR2 VH CDR3, ING1 VLCDR1, ING1 VL CDR2, ING1 VL CDR3, ING1 VH CDR1, ING1 VH CDR2, ING1 VHCDR3, ING2 VL CDR1, ING2 VL CDR2, ING2 VL CDR3, ING2 VH CDR1, ING2 VHCDR2, and ING2 VH CDR3 (see, e.g., FIGS. 18, and 26, Tables 2 and/orTable 13, etc.). In various embodiments the antibodies each comprise aVH CDR1, CDR2, and CDR3 all selected from a VH domain selected from thegroup consisting of a RAZ1 VH domain, a CR1 VH domain, an ING1 VHdomain, an ING2 VH domain, a 1D11 VH domain, a 2G11 VH domain, a 3D12 VHdomain, and a 5G4 VH domain. In various embodiments the antibodies eachcomprise a VL CDR1, CDR2, and CDR3 all selected from a VL domainselected from the group consisting of a RAZ1 VL domain, a CR1 VL domain,a CR2 VL domain, an ING1 VL domain, an ING2 VL domain, a 1D11 VL domain,a 2G11 VL domain, a 3D12 VL domain, and a 5G4 VL domain. In certainembodiments the antibodies each comprise: a VH CDR1, CDR2, and CDR3 allselected from a VH domain selected from the group consisting of a RAZ1VH domain, a CR1 VH domain, a CR2 VII domain, an ING1 VH domain, an ING2VH domain, a 1D11 VH domain, a 2G11 VH domain, a 3D12 VH domain, and a5G4 VH domain; and a VL CDR1, CDR2, and CDR3 all selected from a VLdomain selected from the group consisting of a RAZ1 VL domain, a CR1 VLdomain, a CR2 VL domain, an ING1 VL domain, an ING2 VL domain, a 1D11 VLdomain, a 2G11 VL domain, a 3D12 VL domain, and a 5G4 VL domain. Incertain embodiments at least one of said antibodies comprises a VH CDR1,VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 selected from anantibody selected from the group consisting of RAZ1, CR1, ING1, andING2. In various embodiments at least one of the antibodies is a singlechain Fv (scFv), an IgG, an IgA, an IgM, an Fab, an (Fab′)₂, or an(scFv′)₂. In certain embodiments at least one of said antibodies isselected from the group consisting of RAZ1, CR1, CR2, ING1, ING2, 2G11,3D12, and 5G4.

In various embodiments, this invention provides an isolated antibodythat specifically binds to an epitope specifically bound by an antibodyselected from the group consisting of C25, 1C6, 3D12, B4, 1F3, HuC25,AR1, AR2, AR3, AR4, WR1(V), WR1(T), 3-1, 3-8, 3-10, ING1, CR1, CR2,RAZ1, and/or ING2. In certain embodiments, the antibody binds to andneutralizes one or preferably two or more botulinum neurotoxin subtypes(e.g., BoNT/A1, BoNT/A2, BoNT/A3, etc.). The antibody can be ofvirtually any mammalian animal type (e.g. mouse, human, goat, rabbit) orchimeric (e.g. humanized), but is most preferably human, or humanized.

In one embodiment, the antibody comprises at least one (more preferablyat least two and most preferably at least three) of the variable heavy(V_(H)) complementarity determining regions (CDRs) listed in Table 2,and/or Table 6, and/or Table 9 and/or Table 13 and/or FIG. 26, orconservative substitutions thereof. In another embodiment, the antibodycomprises at least one (more preferably at least two and most preferablyat least three) of the variable light (V_(L)) complementaritydetermining regions (CDRs) listed in Table 2, and/or Table 6, and/orTable 9 and/or Table 13, and/or FIG. 26, or conservative substitutionsthereof. In still another embodiment, the antibody comprises at leastone (more preferably at least two and most preferably at least three) ofthe variable heavy (V_(H)) complementarity determining regions (CDRs)listed in Table 2, and/or Table 6, and/or Table 9 and/or Table 13,and/or FIG. 26, or conservative substitutions thereof and at least one(more preferably at least two and most preferably at least three) of thevariable light (V_(L)) complementarity determining regions (CDRs) listedin Table 2, and/or Table 6, and/or Table 9 and/or Table 13 and/or FIG.26, or conservative substitutions thereof and/or one, two, or three ofthe VL or VH framework regions listed in Table 2, and/or Table 6, and/orTable 9 and/or Table 13, and/or FIG. 26. Certain preferred antibodiesinclude, but are not limited to C25, 1C6, 3D12, B4, 1F3, HuC25, AR1,AR2, AR3, AR4, WR1(V), WR1(T), 3-1, 3-8, 3-10, ING1, CR1, RAZ1, ING2,1D11, 2G11, 3D12, and/or 5G4. Certain preferred antibodies include anIgG, a single chain Fv (scFv), while other preferred antibodies include,but are not limited to an IgG, an IgA, an IgM, a Fab, a (Fab′)₂, a(scFv′)₂, and the like. In certain embodiments, the antibodies can bemulti-valent. The antibodies can include fusion proteins comprising oftwo scFv fragments.

This invention also provides for compositions comprising one or more ofthe botulinum neurotoxin-neutralizing antibodies described herein in apharmacological excipient.

This invention also provides BoNT-neutralizing epitopes. Certainpreferred epitopes include BoNT/A H_(C) epitopes specifically bound byC25, 1C6, 3D12, B4, 1F3, HuC25, AR1, AR2, AR3, AR4, WR1(V), WR1(T), 3-1,3-8, 3-10, ING1, CR1, CR2, RAZ1, ING2, 1D11, 2G11, 3D12, and/or 5G4.Certain preferred polypeptides are not a full-length BoNT and moreparticularly preferred polypeptides are not a full-length BoNT H_(c)fragment. Thus, most preferred epitopes are a BoNT/A H_(C) subsequenceor fragment with preferred subsequences having a length of at least 4,preferably at least 6, more preferably at least 8 and most preferably atleast 10, 12, 14, or even 15 amino acids. In this regard, it is notedthat HuC25 and its derivatives (AR1, 2, 3, 4, and CR1) bind an HC domainthat is N-terminal, while 3D12/RAZ1 bind a HC domain that is C-terminal.Neither of these epitopes are linear.

DEFINITIONS

A “BoNT polypeptide” refers to a Botulinum neurotoxin polypeptide (e.g.,a BoNT/A polypeptide, a BoNT/B polypeptide, a BoNT/C polypeptide, and soforth). The BoNT polypeptide can refer to a full-length polypeptide orto a fragment thereof. Thus, for example, the term “BoNT/A polypeptide”refers to either a full-length BoNT/A (a neurotoxin produced byClostridium botulinum of the type A serotype) or a fragment thereof(e.g. the Hc fragment). The H_(C) fragment approximately a 50 DaC-terminal fragment (residues 873-1296) of BoNT/A (Lacy and Stevens(1999) J. Mol. Biol., 291: 1091-1104).

A “BoNT” serotype refers one of the standard known BoNT serotypes (e.g.BoNT/A, BoNT/C, BoNT/D, BoNT/E, BoNT/F, etc.). BoNT serotypes differfrom each other by as little as about 35% at the amino acid level (e.g.,between BoNT/E and BoNT/F) up to about 66% at the amino acid level,(e.g., for BoNT/A vs BoNT/C or D). Thus, BoNT serotypes differ from eachother by about 35-66% at the amino acid level.

The term “BoNT subtype” (e.g., a BoNT/A1A subtype) refers to botulinumneurotoxin gene sequences of a particular serotype (e.g., A, C, D, F,etc.) that differ from each other sufficiently to produce differentialantibody binding. In certain embodiments, the subtypes differ from eachother by at least 2.5%, preferably by at least 5%, or 10%, morepreferably by at least 15% or 20% at the amino acid level. In certainembodiments, the subtypes differ from each other by nor more than 35%,preferably by no more than 31.6%, still more preferably by no more than30%, or 25%, more preferably by less than about 20% or 16% at the aminoacid level. In certain embodiments, BoNT subtypes differ from each otherby at least 2.6%, more preferably by at least 3%, and most preferably byat least 3.6% at the amino acid level. BoNT subtypes typically differfrom each other by less than about 31.6%, more preferably by less thanabout 16%, at the amino acid level.

“Neutralization” refers to a measurable decrease in the toxicity of aBotulinum neurotoxin (e.g., BoNT/A).

The term “high affinity” when used with respect to an antibody refers toan antibody that specifically binds to its target(s) with an affinity(K_(D)) of at least about 10⁻⁸ M, preferably at least about 10⁻⁹ M, morepreferably at least about 10⁻¹⁰ M, and most preferably at last about10⁻¹¹ M. In certain embodiments “high affinity” antibodies have a K_(D)that ranges from about 1 nM to about 5 pM.

The following abbreviations are used herein: AMP, ampicillin; BIG,botulinum immune globulin; BoNT, botulinum neurotoxin; BoNT/A, BoNT typeA; CDR, complementarity determining region; ELISA, enzyme-linkedimmunosorbent assay; GLU, glucose; HBS, HEPES-buffered saline (10 mMHEPES, 150 mM NaCl [pH 7.4]); H_(c), c-terminal domain of BoNT heavychain (binding domain); H_(N), N-terminal domain of BoNT heavy chain(translocation domain); IgG, immunoglobutin G; IMAC, immobilized-metalaffinity chromatography; IPTG, isopropyl-β-D-thiogalactopyranoside; KAN,kanamycin; K_(d), equilibrium constant; k_(off), dissociation rateconstant; k_(on), association rate constant; MPBS, skim milk powder inPBS; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline (25 mMNaH₂PO₄, 125 mM NaCl [pH 7.0]; RU, resonance units; scFv, single-chainFv antibody fragments; TPBS, 0.05% (vol/vol) Tween 20 in PBS; TMPBS,0.05% (vol/vol) Tween 20 in MPBS; TU, transducing units; V_(H),immunoglobulin heavy-chain variable region; V_(K), immunoglobulin kappalight-chain variable region; V_(L) immunoglobulin light-chain variableregion; wt, wild type.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are preferably in the natural “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR (1.822(b)(4).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include Fab′₂, IgG,IgM, IgA, and single chain antibodies, more preferably single chain Fv(scFv) antibodies in which a variable heavy and a variable light chainare joined together (directly or through a peptide linker) to form acontinuous polypeptide.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

An S25 antibody refers to an antibody expressed by clone S25 or to anantibody synthesized in other manners, but having the same CDRs andpreferably, but not necessarily, the same framework regions as theantibody expressed by clone s25. Similarly, antibodies C25, 1C6, 3D12,B4, 1F3, HuC25, AR1, AR2, AR3, AR4, WR1(V), WR1(T), 3-1, 3-8, 3-10,ING1, CR1, RAZ1, or ING2 refer to antibodies expressed by thecorresponding clone(s) and/or to antibodies synthesized in othermanners, but having the same CDRs and preferably, but not necessarily,the same framework regions as the referenced antibodies.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smaller Kdrepresents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. Ann. Rev. Biochem., 59: 439-473(1990).

A “BoNT-neutralizing antibody” refers to an antibody that binds to oneor more Botulinum neurotoxin(s) (e.g., BoNT/A1, BoNT/A2, etc.) and thatby so-binding reduces the toxicity of that BoNT neurotoxin. Thus, forexample the term “BoNT/A-neutralizing antibody”, as used herein refersto an antibody that specifically binds to a BoNT/A polypeptide (e.g. aBoNT/A1 polypeptide), in certain embodiments, to an H_(C) domain of aBoNT/A polypeptide and that by so-binding reduces the toxicity of theBoNT/A polypeptide. Reduced toxicity can be measured as an increase inthe time that paralysis developed and/or as a lethal dosage (e.g. LD₅₀)as described herein. Antibodies derived from BoNT-neutralizingantibodies include, but are not limited to, the antibodies whosesequence is expressly provided herein.

Antibodies derived from BoNT-neutralizing antibodies preferably have abinding affinity of about 1.6×10⁻⁸ or better and can be derived byscreening libraries of single chain Fv fragments displayed on phage oryeast constructed from heavy (VH) and light (VL) chain variable regiongenes obtained from mammals, including mice and humans, immunized withbotulinum toxoid, toxin, or BoNT fragments. Antibodies can also bederived by screening phage or yeast display libraries in which a knownBoNT-neutralizing variable heavy (V_(H)) chain is expressed incombination with a multiplicity of variable light (V_(L)) chains orconversely a known BoNT-neutralizing variable light chain is expressedin combination with a multiplicity of variable heavy (V_(H)) chains.BoNT-neutralizing antibodies also include those antibodies produced bythe introduction of mutations into the variable heavy or variable lightcomplementarity determining regions (CDR1, CDR2 or CDR3) as describedherein. Finally BoNT-neutralizing antibodies include those antibodiesproduced by any combination of these modification methods as applied tothe BoNT-neutralizing antibodies described herein and their derivatives.

A neutralizing epitope refers to the epitope specifically bound by aneutralizing antibody.

A single chain Fv (“scFv” or “scFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which may be expressed from a nucleic acidincluding V_(H)- and V_(L)-encoding sequences either joined directly orjoined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat.Acad. Sci. USA, 85: 5879-5883. A number of structures for converting thenaturally aggregated—but chemically separated light and heavypolypeptide chains from an antibody V region into an scFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g. U.S. Pat. Nos.5,091,513 and 5,132,405 and 4,956,778.

In one class of embodiments, recombinant design methods can be used todevelop suitable chemical structures (linkers) for converting twonaturally associated—but chemically separate—heavy and light polypeptidechains from an antibody variable region into a scFv molecule which willfold into a three-dimensional structure that is substantially similar tonative antibody structure.

Design criteria include determination of the appropriate length to spanthe distance between the C-terminal of one chain and the N-terminal ofthe other, wherein the linker is generally formed from small hydrophilicamino acid residues that do not tend to coil or form secondarystructures. Such methods have been described in the art. See, e.g., U.S.Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No.4,946,778 to Ladner et al.

In this regard, the first general step of linker design involvesidentification of plausible sites to be linked. Appropriate linkagesites on each of the V_(H) and V_(L) polypeptide domains include thosewhich will result in the minimum loss of residues from the polypeptidedomains, and which will necessitate a linker comprising a minimum numberof residues consistent with the need for molecule stability. A pair ofsites defines a “gap” to be linked. Linkers connecting the C-terminus ofone domain to the N-terminus of the next generally comprise hydrophilicamino acids which assume an unstructured configuration in physiologicalsolutions and preferably are free of residues having large side groupswhich might interfere with proper folding of the V_(H) and V_(L) chains.Thus, suitable linkers under the invention generally comprisepolypeptide chains of alternating sets of glycine and serine residues,and may include glutamic acid and lysine residues inserted to enhancesolubility. One particular linker under the invention has the amino acidsequence [(Gly)₄Ser]₃ (SEQ ID NO:1). Another particularly preferredlinker has the amino acid sequence comprising 2 or 3 repeats of[(Ser)₄Gly] (SEQ ID NO:2), such as [(Ser)₄Gly]₃ (SEQ ID NO:3), and thelike. Nucleotide sequences encoding such linker moieties can be readilyprovided using various oligonucleotide synthesis techniques known in theart. See, e.g., Sambrook, supra.

The phrase “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. Specific binding to a proteinunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, BoNT/A-neutralizingantibodies can be raised to BoNT/A protein)s that specifically bind toBoNT/A protein(s), and not to other proteins present in a tissue sample.A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the strategy for in vitro antibody production usingphage libraries. mRNA is prepared from splenocytes, first-strand cDNA isprepared, and antibody V_(H) and V_(L) genes are amplified by PCR. V_(H)and V_(L) genes are spliced together randomly using PCR to create arepertoire of scFv genes. The scFv gene repertoire is cloned into aphagemid vector in frame with a gene (gIII) encoding a phagemid minorcoat protein (pIII). Each phage in the resulting phage antibody libraryexpresses and scFv-pIII fusion protein on its surface and contains thegene encoding the scFv inside. Phage antibodies binding a specificantigen can be separated from nonbinding phage antibodies by affinitychromatography on immobilized antigen. A single round of selectionincreases the number of antigen-binding phage antibodies by a factorranging from 20 to 10,000 depending on the affinity of the antibody.Eluted phage antibodies are used to infect E. coli, which then producemore phage antibodies for the next round of selection. Repeated roundsof selection make it possible to isolate antigen-binding phageantibodies that were originally present at frequencies of less than onein a billion.

FIG. 2 panel A and panel B show sensor grams illustrating the techniqueused to epitope map scFv binding to BoNT/A H_(C). Epitope mapping wasperformed by using surface plasmon resonance in a BIAcore, with scFvstudied in pairs. Each scFv was injected into the BIAcore and allowed tobind to BoNT/A H_(C) coupled to the sensor chip surface until saturationwas achieved. The amount (in RU) bound for each scFv alone was comparedto the amount bound when the two scFv were mixed and injected together.Point a shows the baseline, followed by the beginning of injection.Points b₁ and b₂ show the initial association phase. Points c₁ and c₂show the beginning of dissociation. The differences in RU between pointsa and c equal the amount of scFv bound to BoNT/A H_(C). Panel A showstwo scFv recognizing different epitopes (C25 and C9). The amount boundof the two scFv injected together (C9/C25, point c₂) is the sum of thetwo scFv injected alone (c₁). Panel B shows two scFv recognizing thesame epitope (C39 and C25). The amount bound for the two scFv injectedtogether (C25/C39; point c) is the same as that for the two scFvinjected alone (c). The large differences in RU between points b₁ andc₁, b₂ and c₂, and b₁ and c are due to differences in refractive indexbetween scFv and running buffer.

FIG. 3 shows the evaluation of scFv neutralization of BoNT/A in a mousehemidiaphragm model. The twitch tension developed after electricalstimulation of a mouse hemidiaphragm was measured below (−30 to 0 min)and after the addition of 20 pM BoNT/A (control), 20 pM BoNT/A plus 20nM scFv S25, C25, 1C6, or 1F3 (representing epitopes 1 to 4respectively), or a combination of S25 and C25 at a final concentrationof 20 nM each. Results are expressed as the fraction of steady-statetwitch tension (at 0 min) versus time. scFv 1C6 and 1F3 do not alter thetime to 50% twitch reduction, whereas scFv C25 and S25 significantlyprolong it. The combination of S25 and C25 significantly prolonged thetime to neuroparalysis compared to C25 or S25 alone.

FIG. 4 shows in vitro toxin neutralization by mAb, pairs of mAbs, andoligoclonal Ab. Time to 50% twitch reduction was measured in isolatedmouse hemidiaphragms and reported for toxin only control, single mAb(C25, S25, or 3D12), pairs of mAbs (C25 S25, C25+3D12, or 3D12+S25), andoligoclonal Ab (C25+3D12+S25). Single mAb significantly prolonged timeto neuroparalysis compared with toxin only. Pairs of mAbs significantlyprolonged time to neuroparalysis compared with single mAbs.

FIGS. 5A and 5B show in vivo toxin neutralization by mAbs (FIG. 5A) andpairs (FIG. 5B) of mAbs. Fifty micrograms total Ab was mixed with 20 or100 mouse LD₅₀s of toxin and injected i.p. Time to death and number ofsurviving mice was determined. No single mAb showed significantprotection against 20 LD₅₀s. All mice survived challenge with 100 LD₅₀swhen given any pair of mAbs.

FIG. 6 shows in vivo toxin neutralization by mAbs, pairs of mAbs, andoligoclonal Ab. In vivo toxin neutralization was determined for mAbs,pairs of mAbs, and oligoclonal Ab at increasing toxin challenge doses.No single mAb showed significant protection. In contrast all mAb pairsneutralized at least 100 LD₅₀s, with approximately 50% of mice survivingchallenge with 1,500 LD₅₀s of toxin for the most potent pair (C25+3D12).Oligoclonal Ab was even more potent with approximately 50% of micesurviving challenge with 20,000 LD₅₀s of toxin.

FIG. 7 shows solution equilibrium dissociation constants (K_(d)) ofantibodies. The solution K_(d) of single mAb C²⁵ and 3D12 weredetermined in a flow fluorimeter by measuring the amount of free Abpresent as a function of increasing BoNT H_(C) toxin. Combining C25 and3D12 mAb in equimolar amounts decreased the C25 K_(d) more than100-fold. Adding a third Ab (S25) decreased the Kd another 4-fold to 18pM.

FIGS. 8A and 8B show ELISA characterization of soluble scFv antibodies.Assays were performed by immobilizing each indicated BoNT serotype,BoNT/A HC and BoNT/A HN coated onto a polystyrene plate. FIG. 8A:Bacterially expressed scFv antibodies derived from the immune library,reactive with the coated antigen was detected with theperoxidase-conjugated mAb anti-E antibody (1:2500). FIG. 8B: Bacteriallyexpressed scFv antibodies derived from the non-immune library, reactivewith the coated antigen were detected with 9E10 antibody (1:500)followed by peroxidase-conjugated anti-mouse-Fc antibody. The results ofthe assay are shown as absorbance at 405 nm which have not beennormalized for protein concentrations.

FIGS. 9A and 9B show sensorgrams of epitope mapping of scFv binding toBoNT/A H_(C). Point ‘a’: beginning of injection, point ‘b’: end ofinjection, and point ‘c’: amount of scFv bound. The difference in RUbetween points b and c is due to differences in refractive index betweenscFv and running buffer. FIG. 9A: The scFv 3A6 and 3D12 recognize thesame epitope, as indicated by no increase in the RU bound when the twoscFv are mixed. FIG. 9B: scFv 2A9 and 2A1 recognize different epitopes,as indicated by an almost additive increase in the RU bound when the twoscFv are mixed.

FIGS. 10A and 10B show the individual and combined effects of scFvantibodies targeting BoNT/A HC domain. FIG. 10A: The twitch tensiondeveloped after electrical stimulation of a mouse hemidiaphragm wasmeasured before (−30 to 0 min) and after the addition of 20 pM BoNT/A(control), 20 pM BoNT/A plus 20 nM of members of cluster I (3D12),cluster II (3F10), C25 or S25. The scFv 3F10 did not alter the time to50% twitch reduction, whereas scFv C25, S25 or 3D12 significantlyprolong the time to 50% twitch reduction. FIG. 10B: The combination ofC25 with S25 or 3D12 (cluster I) prolong significantly the time to 50%twitch reduction.

FIG. 11 shows a phylogenetic tree of published botulinum neurotoxingenes. The phylogenetic tree was constructed from the DNA sequences ofpublished Clostridial neurotoxin genes using Vector NTI software.

FIGS. 12A and 12B show an analysis of BoNT/A gene sequences. FIG. 12A:Phylogenetic tree of BoNT/A genes reveals two clusters, A1 and A2. FIG.12B: Model of the amino acid side chain differences between BoNT/A1 andBoNT/A2. The BoNT/A heavy chain binding domain is in white at the top ofthe figure, with the putative ganglioside binding residues in blue andthe ganglioside in red. The heavy chain translocation domain is inorange and the light chain in white at the bottom of the figure. Sidechain differences between BoNT A1 and A2 toxins are shown in green.

FIG. 13 shows an analysis of BoNT/B gene sequences. A phylogenetic treeof BoNT/B genes reveals four clusters: BoNT/B1, BoNT/B2, nonproteolyticBoNT/B, and bivalent BoNT/B. Percent differences between clusters rangefrom 3.6 to 7.7%. As with BoNT/A, the greatest differences are seen inthe heavy chain.

FIG. 14 shows binding of BoNT/A H_(C) monoclonal antibodies (C25, B4,S25, and 3D12) to BoNT/A1 and BoNT/A2 toxins as determined by captureELISA. Wells were coated with the indicated mAb followed by varyingconcentrations of pure or complex BoNT/A1 or BoNT/A2. Toxin binding wasdetected using polyclonal equine BoNT/A antisera. A1 toxins areindicated by solid squares; A2 toxins by open circles. Pure toxins aresolid lines; toxin complexes are dashed lines.

FIG. 15 shows binding of BoNT/A translocation domain and light chainmonoclonal antibodies to BoNT/A1 and BoNT/A2 toxins as determined bycapture ELISA. Methods were as described for FIG. 14.

FIG. 16 illustrates the ability of mAb pairs to protect mice challengedwith BoNT/A1 toxin. A range of mouse LD₅₀s of BoNT/A1 toxin complex wasmixed with 50 ug of an equimolar ratio of the indicated mAbs and themixture was injected intraperitoneally. The number of mice surviving vschallenge dose is indicated.

FIGS. 17A and 17B illustrate the ability of mAb triplets to protect micechallenged with BoNT/A1 or BoNT/A2 toxins. A range of mouse LD₅₀s ofBoNT/A1 toxin complex (FIG. 17A) or BoNT/A2 toxin complex (FIG. 17B) wasmixed with 50 ug of an equimolar ratio of the indicated mAbs and themixture was injected intraperitoneally. The number of mice survivingversus challenge dose is indicated.

FIG. 18. Sequences for mutated and selected antibodies (HU-C25 (V_(H)SEQ ID NO:4, V_(L) SEQ ID NO: 17), AR1 (V_(H) SEQ ID NO:5, V_(L) SEQ TDNO: 18), AR2 (V_(H) SEQ ID NO:6, V_(L) SEQ ID NO: 19), AR3 (V_(H) SEQ IDNO:7, V_(L) SEQ ID NO: 20), AR4 (V_(H) SEQ ID NO:8, V_(L) SEQ ID NO:21), CR1 (V_(H) SEQ ID NO:9, V_(L) SEQ ID NO: 22)). Dashes indicateconserved residues. Letters indicate mutated residues. See also SEQ IDNOs:363-366 (CR1).

FIGS. 19A and 19B. Sequences for mutated and selected antibodies. FIG.19A: 3D12 (V_(H) SEQ ID NO: 11, V_(L) SEQ ID NO: 10), and RAZ1 (V_(H)SEQ ID NO:11, V_(L) SEQ ID NO: 354)). FIG. 19B: ING1 (V_(H) SEQ IDNO:12, V_(L) SEQ ID NO: 358), 1D11 (V_(H) SEQ TD NO: 12, V_(L) SEQ IDNO: 13), 2G11 (V_(H) SEQ ID NO: 12, V_(L) SEQ ID NO: 14), 5G4 (V_(H) SEQID NO: 12, V_(L) SEQ ID NO: 15), ING2 (V_(H) SEQ ID NO: 16, V_(L) SEQ TDNO: 362). Dashes indicate conserved residues. Letters indicate mutatedresidues. See also SEQ ID NOs:351-354 (RAZ1), SEQ ID NOs:355-358 (ING1),SEQ ID NOs: 427-430 359-362 (ING2).

FIGS. 20A and 20B show a scheme used for affinity maturation of HuC25(FIG. 20A) and 3D12 (FIG. 20B) scFv using yeast display.

FIGS. 21A through 21D show affinities of wild type and affinity maturedyeast displayed scFv. FIG. 21A: Hu C25 and AR1; FIG. 21B: AR1 and AR2;FIG. 21C: AR2 and AR4; FIG. 21D: 3D12 and RAZ1.

FIG. 22 illustrates detection of BoNT/A by flow cytometry using wildtype and affinity matured antibodies.

FIGS. 23A and 23B show the potency of neutralization of BoNT/A by wildtype and affinity matured antibodies. FIG. 23A: 100 mouse LD50challenge. FIG. 23B: 200 mouse LD50 challenge.

FIGS. 24A 24B show potency of neutralization of BoNT/A by pairs of wildtype and affinity matured antibodies. FIG. 24A: 500 mouse LD₅₀challenge. FIG. 24B: 5000 mouse LD₅₀ challenge.

FIG. 25 illustrates neutralization of BoNT/A2 by antibody combinations.

FIG. 26 shows alignment of Hu-C25 lineage antibodies (HU-C25 (V_(H) SEQID NO:4, V_(L) SEQ ID NO:17), AR1 (V_(H) SEQ ID NO:5, V_(L) SEQ IDNO:18), AR2 (V_(H) SEQ ID NO:6, V_(L) SEQ ID NO:19), AR3 (V_(H) SEQ IDNO:7, V_(L) SEQ ID NO:20), AR4 (V_(H) SEQ ID NO:8, V_(L) SEQ ID NO:21),CR1 (V_(H) SEQ ID NO:9, V_(L) SEQ ID NO:22), and CR2 (V_(H) SEQ IDNO:23, V_(L) SEQ ID NO: 22)). See also SEQ ID NOs:367-372 (CR2).

DETAILED DESCRIPTION

This invention provides novel antibodies that specifically bind to andneutralize botulinum neurotoxin type A and, in certain embodiments,other botulinum neurotoxin serotypes (e.g., B, C, D, E, F, etc.).Botulinum neurotoxin is produced by the anaerobic bacterium Clostridiumbotulinum. Botulinum neurotoxin poisoning (botulism) arises in a numberof contexts including, but not limited to food poisoning (food bornebotulism), infected wounds (wound botulism), and “infant botulism” fromingestion of spores and production of toxin in the intestine of infants.Botulism is a paralytic disease that typically begins with cranial nerveinvolvement and progresses caudally to involve the extremities. In acutecases, botulism can prove fatal.

Botulism neurotoxins (BoNTs) are also classified by the Centers forDisease Control (CDC) as one of the six highest-risk threat agents forbioterrorism (the “Category A agents”), due to their extreme potency andlethality, ease of production and transport, and the need for prolongedintensive care (Arnon et al. (2001) JAMA 285: 1059-1070). Both Iraq andthe former Soviet Union produced BoNT for use as weapons (UN SecurityCouncil (1995) supra; Bozheyeva (1999) supra.) and the Japanese cult AumShinrikyo attempted to use BoNT for bioterrorism (Arnon (2001) supra.).As a result of these threats, specific pharmaceutical agents are neededfor prevention and treatment of intoxication.

It has recently been discovered that there are multiple subtypes ofvarious BoNT serotypes. Moreover, we have further discovered that manyantibodies that bind, for example the BoNT/A1 subtype will not bind theBoNT/A2 subtype, and so forth

We have discovered that particularly efficient neutralization of abotulism neurotoxin (BoNT) subtype is achieved by the use ofneutralizing antibodies that bind two or more subtypes of the particularBoNT serotype with high affinity. While this can be accomplished byusing two or more different antibodies directed against each of thesubtypes, this is less effective, inefficient and not practical. A BoNTtherapeutic is desirably highly potent, given the high toxicity of BoNT.Since it is already necessary to use multiple antibodies to neutralize agiven BoNT serotype with the desired potency (see below and FIGS. 5, 6,16, and 17), the number of antibodies required would be prohibitive froma manufacturing standpoint if it were necessary to use differentantibodies for each subtype. Increasing the number of antibodies in themixture also reduces the potency. Thus, for example, if in a mixture offour antibodies, two neutralize A1 and two neutralize A2 toxin, thenonly 50% of the antibody will neutralize a given toxin. In contrast amixture of two antibodies both of which neutralize A1 and A2 toxins willhave 100% activity against either toxin and will be simpler tomanufacture. For example for two BoNT/A subtypes (A1, A2) potentneutralization can be achieved with two to three antibodies. Ifdifferent antibodies were required for BoNT/A1 and BoNT/A2neutralization, then four to six antibodies would be required. Thecomplexity increases further for additional subtypes. Thus, in certainembodiments this invention provides for neutralizing antibodies thatbind two or more BoNT subtypes (e.g., BoNT/A1, BoNT/A2, etc.) with highaffinity.

Examples of antibodies that bind both BoNT/A1 and BoNT/A2 with highaffinity include, but are not limited to, CR1, RAZ1, ING1, and ING2described herein.

It was also a surprising discovery that when one starts combiningneutralizing antibodies that the potency of the antibody combinationincreases dramatically. This increase makes it possible to generate abotulinum antibody of the required potency for therapeutic use. It wasalso surprising that as one begins combining two and three monoclonalantibodies, the particular BoNT epitope that is recognized becomes lessimportant. Thus for example, as indicated in Example 5, antibodies thatbind to the translocation domain and/or catalytic domains of BoNT hadneutralizing activity, either when combined with each other or whencombined with a mAb recognizing the BoNT receptor binding domain (HC)were effective in neutralizing BoNT activity. Thus, in certainembodiments, this invention contemplates compositions comprising atleast two, more preferably at least three high affinity antibodies thatbind non-overlapping epitopes on the BoNT.

Thus, in certain embodiments, this invention contemplates compositionscomprising two or more, preferably three or more different antibodiesselected from the group consisting of 3D12, RAZ1, CR1, ING1, ING2, an/orantibodies comprising one or more CDRs from these antibodies, and/or oneor more antibodies comprising mutants of these antibodies, such as the1D11, 2G11, or 5G4 mutants of ING1 (see, e.g., FIG. 19B).

As indicated above, in certain embodiments, the antibodies provided bythis invention bind to and neutralize one or more botulinum neurotoxintype A (BoNT/A) subtypes. Neutralization, in this context, refers to ameasurable decrease in the toxicity of BoNT/A. Such a decrease intoxicity can be measured in vitro by a number of methods well known tothose of skill in the art. One such assay involves measuring the time toa given percentage (e.g. 50%) twitch tension reduction in ahemidiaphragm preparation. Toxicity can be determined in vivo, e.g. asan LD₅₀ in a test animal (e.g. mouse) botulinum neurotoxin type A in thepresence of one or more putative neutralizing antibodies. Theneutralizing antibody can be combined with the botulinum neurotoxinprior to administration, or the animal can be administered the antibodyprior to, simultaneous with, or after administration of the neurotoxin.

As the antibodies of this invention act to neutralize botulinumneurotoxin type A, they are useful in the treatment of pathologiesassociated with botulinum neurotoxin poisoning. The treatmentsessentially comprise administering to the poisoned organism (e.g. humanor non-human mammal) a quantity of one or more neutralizing antibodiessufficient to neutralize (e.g. mitigate or eliminate) symptoms of BoNTpoisoning.

Such treatments are most desired and efficacious in acute cases (e.g.where vital capacity is less than 30-40 percent of predicted and/orparalysis is progressing rapidly and/or hypoxemia with absolute orrelative hypercarbia is present. These antibodies can also be used totreat early cases with symptoms milder than indicated (to preventprogression) or even prophylactically (a use the military envisions forsoldiers going in harms way). Treatment with the neutralizing antibodycan be provided as an adjunct to other therapies (e.g. antibiotictreatment).

The antibodies provided by this invention can also be used for the rapiddetection/diagnosis of botulism (type A toxin(s)) and thereby supplementand/or replace previous laboratory diagnostics.

In another embodiment this invention provides the epitopes specificallybound by botulinum neurotoxin type A neutralizing antibodies. Theseepitopes can be used to isolate, and/or identify and/or screen for otherantibodies BoNT/A neutralizing antibodies as described herein.

I. Potency of Botulinum Neurotoxin (BoNT)-Neutralizing Antibodies

Without being bound to a particular theory, it is believed that thecurrent antitoxins used to treat botulism (horse and human) have apotency of about 5000 mouse LD50s/mg (human) and 55,000 mouse LD50s mg(horse).

Based on our calculations, we believe a commercially desirable antitoxinwill have a have a potency greater than about 10,000 to 100,000LD50s/mg. Combinations of the antibodies described herein (e.g., two orthree antibodies) meet this potency. Thus, in certain embodiments, thisinvention provides antibodies and/or antibody combinations thatneutralize at least about 10,000 mouse LD50s/mg of antibody, preferablyat least about 15,000 mouse LD50s/mg of antibody, more preferably atleast about 20,000 mouse LD50s/mg of antibody, and most preferably atleast about 25,000 mouse LD50s/mg of antibody.

II. Botulinum Neurotoxin (BoNT)-Neutralizing Antibodies.

In certain preferred embodiments, BoNT neutralizing antibodies areselected that bind to one, but more preferably, to at least two or BoNTsubtypes. A number of subtypes are known for each BoNT serotype. Thus,for example, BoNT/A subtypes include, but are not limited to, BoNT/A1,BoNT/A2, BoNT/A3, and the like (see, e.g., FIG. 11). It is also noted,for example, that the BoNT/A1 subtype includes, but is not limited to62A, NCTC 2916, ATCC 3502, and Hall hyper (Hall Allergan) and areidentical (99.9-100% identity at the amino acid level.) and have beenclassified as subtype A1 (FIG. 12A). The BoNT/A2 sequences (Kyoto-F andFRI-A2H) (Willems, et al. (1993) Res. Microbiol. 144:547-556) are 100%identical at the amino acid level. Another BoNT/A subtype, (that we arecalling A3) is produced by a strain called Loch Maree that killed anumber of people in an outbreak in Scotland. We have data that three ofantibodies described herein that cross react with both A1 and A2 toxins(see Table 1) also cross react with A3 toxin (these would be CR1, ING1,and RAZ1). Another BoNT/A toxin we have identified we refer to as A4. Itis produced by a bivalent Clostridial strain that produces both B and Atoxins.

Similarly, as shown in FIG. 11, a number of subtypes are also known forserotypes B, C, E, and F. Using, the methods described herein, it wasdiscovered that high-affinity antibodies that are cross-reactive withtwo or more subtypes within a serotype can be produced (e.g.,selected/engineered). Moreover, without being bound to a particulartheory, it appears that these cross-reactive antibodies aresubstantially more efficient in neutralizing Botulinum neurotoxin,particularly when used in combination one or more different neutralizingantibodies.

The sequences of the variable heavy (VH) and variable light (VL) domainsfor a number of prototypically “cross-reactive” antibodies areillustrated in Table 2 and in FIGS. 18 and 19. As indicated above, theantibodies CR1, RAZ1, ING1, and ING2 are cross-reactive for the BoNT/A1and BoNT/A2 subtypes, while the antibodies CR1, ING1, and RAZ1 areadditionally cross-reactive for the BoNT/A3 subtype.

The antibody CR1 was produced by the mutation and selection of humanizedC25 (HuC25), a derivative of AR2, e.g., as described in Example 4. Theantibody was mutated and selected on both the A1 and A2 subtypes.Similarly mutation of the antibody 3D12 (see, e.g., Example 2) yieldedRAZ1. Selection of immune scFv libraries on yeast yielded ING1 and ING2.

TABLE 1 Binding data for engineered antibodies. KD BoNT/A1 KD BoNT/A2mAb (×10⁻¹²M) (×10⁻¹²M) HuC25 45 >100,000 AR2 7.2 >100,000 CR1 6.2 17003D12 61 152 RAZ1 1.7 3.7 B4 96 No binding ING1 560 750 ING2 16.7 15.4

Table 2 and FIGS. 18 AND 19 provide amino acid sequence information forthe VH and VL regions of the cross-reactive antibodies RAZ1, CR1, ING1,and ING2. Similar information is provided for the antibodies AR2 and AR3which specifically bind to the BoNT/A1 subtype. In addition sequenceinformation is provided herein for S25, C25, C39, 1C6, 3D12, B4, 1F3,HuC25, AR1, AR2, WR1(V), WR1(T), 3-1, 3-8, and/or 3-10 (see, e.g., Table6, and/or Table 11 and/or Table 13).

TABLE 2 Amino acid sequences for affinity matured, cross reactive,and/or modified antibodies. Clone Framework 1 CDR1 Framework 2 CDR2Heavy Chains AR3 QVQLQESGGGLVQPGGSLRLSC EHYMY (SEQ WVRQAPGKGLEWTISDGGSYTYYPD AASGFTFG (SEQ ID ID NO:25) VA (SEQ ID SVEG (SEQ ID NO:24)NO:26) NO:27) AR4 QVQLQESGGGLVQPGGSLRLSC EHYMY (SEQ WVRQAPGKGLEWTISDGGSYTYYPD AASGFTFE (SEQ ID ID NO:29) VA (SEQ ID SVEG (SEQ ID NO:28)NO:30) NO:31) CR1 QVQLQESGGGLVQPGGSLRLSC YDYMY (SEQ WVRQAPGKGLEWTISDGGSYTYYSD AASGFTFK (SEQ ID ID NO:33) VA (SEQ ID SVEG (SEQ ID NO:32)NO:34) NO:35) CR2 RAZ1 QVQLVQSGGGVVHPGRSLKLSC DYDMH (SEQ WVRQAPGKGLEWVMWFDGTEKYSAE AGSGFTFS (SEQ ID ID NO:37) VA (SEQ ID SVKG (SEQ ID NO:36)NO:38) NO:39) ING1 QVQLQQSGGGLVQPGGSLRLSC NYAMT (SEQ WVRQAPGKGLEWSISVGGSDTYYAD AASGFTFS (SEQ ID NO:40) ID NO:41) VS (SEQ ID SVKG (SEQ IDNO:42) NO:43) ING2 QVQLVQSGAEVKKPGSSVKVSC RNAIA (SEQ WVRQAPGQGLEWRIIPNLRTTHYAQ KASGDTFN (SEQ ID ID NO:45) MG (SEQ ID KFQG (SEQ ID NO:44)NO:46) NO:47) 2G11 QVQLQQSGGGLVQPGGSLRLSC NYAMT (SEQ WVRQAPGKGLEWSISVGGSDTYYAD AASGFTFS (SEQ ID ID NO:49) VS (SEQ ID SVKG (SEQ ID NO:48)NO:50) NO:51) 5G4 QVQLQQSGGGLVQPGGSLRLSC NYAMT (SEQ WVRQAPGKGLEWSISVGGSDTYYAD AASGFTFS (SEQ ID ID NO:53) VS (SEQ ID SVKG (SEQ ID NO:52)NO:54) NO:55) Framework 3 CDR3 Framework 4 AR3 RFTTSRDNSKNTLYLQMNSLRAYRYDDAMDY WGQGTLVTVSS EDTAIYYCSR (SEQ ID (SEQ ID (SEQ ID NO:56) NO:57)NO:58) AR4 RFTTSRDNSKNTLYLQMNSLRA YRYDDAMDY WGQGTLVTVSS EDTAIYYCSR (SEQID (SEQ ID (SEQ ID NO:59) NO:60) NO:61) CR1 RFTTSRDNSKNTLYLQMNSLRAYRYDDAMDY WGQGTRVTVSS EDTAIYYCSR (SEQ ID (SEQ ID (SEQ ID NO:62) NO:63)NO:64) CR2 RAZ1 RFTISRDNSKNTLFLQMNSLRA EPDWLLWGDRG WGQGTTVTVSSDDTAVYYCAR (SEQ ID ALDV (SEQ (SEQ ID NO:65) ID NO:66) NO:67) ING1RFTVSRDNSKNTLLLQMNSLRA VRTKYCSSLSC WGQGTLVTVSS EDTAVYYCAK (SEQ ID FAGFDS(SEQ (SEQ ID NO:68) IDNO:69) NO:70) ING2 RVAITADKHTNTVFMELSSLRSDPYYYSYMDV WGKGTTVTVSS EDTAVYYCAR (SEQ ID (SEQ ID (SEQ ID NO:71) NO:72)NO:73) 2G11 RFTVSRDNSKNTLLLQMNSLRA VRTKYCSSLSC WGQGTRVTVSS EDTAVYYCAK(SEQ ID FAGFDS (SEQ (SEQ ID NO:74) ID NO:75) NO:76) 5G4RFTVSRDNSKNTLLLQMNSLRA VRTKYCSSLSC WGQGTRVTVSS EDTAVYYCAK (SEQ ID FAGFDS(SEQ (SEQ ID NO:77) ID NO:78) NO:79) Clone Framework 1 CDR1 Framework 2CDR2 Light Chains: AR3 EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRLRASNLEP (SEQ C (SEQ ID NO:80) SFMQ (SEQ LIY (SEQ ID ID NO:83) ID NO:81)NO:82) AR4 EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQC (SEQ ID NO:84) SFMQ (SEQ LIY (SEQ ID ID NO:87) ID NO:85) NO:86) CR1EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQ C (SEQ IDNO:88) SFMQ (SEQ LIY (SEQ ID ID NO:91) ID NO:89) NO:90) RAZ1DIVMTQSPSTLSASVGDRVTIT WASQSISSRLA WYQQKPGKAPKL EATSLGS (SEQ C (SEQ IDNO:92) (SEQ ID LMY (SEQ ID ID NO:95) NO:93) NO:94) ING1DIVMTQSPSSLSASVGDRVTIT RASQSISSYLN WYQQKPGKAPKL AASSLQS (SEQ C (SEQ IDNO:96) (SEQ ID LIY (SEQ ID ID NO:99) NO:97) NO:98) ING2EIVLTQSPDSLAVSLGERATIN KSSRSVLYSSN WYQQKPGQPPKL WASTRES (SEQ C (SEQ IDNO:100) NNNYLA (SEQ LIY (SEQ ID ID NO:103) ID NO:101) NO:102) 2G11DVVMTQSPSSLSASVGDRVTIT RASQSISSYLH WYQQKPGKAPTL DASSSQS (SEQ C (SEQ IDNO:104) (SEQ ID LIS (SEQ ID ID NO:107) NO:105) NO:106) 5G4EIVLTQSPSSLSASVGDRVTIT RASQGISNYLA WYQQKPGKVPKL AASTLQS (SEQ C (SEQ IDNO:108) (SEQ ID LIY (SEQ ID ID NO:111) NO:109) NO:110) Clone Framework 3CDR3 Framework 4 AR3 GIPARFSGSGSGTDFTLTISSL QQGNEVPFT FGQGTKVEIKREPEDFAVYYC (SEQ ID (SEQ ID (SEQ ID NO:112) NO:113) NO:114) AR4GIPARFSGSGSGTDFTLTISSL QQGNEVPFT FGQGTKVEIKR EPEDFAVYYC (SEQ ID (SEQ ID(SEQ ID NO:115) NO:116) NO:117) CR1 GIPARFSGSGSGTDFTLTISSL QQGNEVPFTFGQGTKVEIKR EPEDFAVYYC (SEQ ID (SEQ ID (SEQ ID NO:118) NO:119) NO:120)RAZ1 GVPSRFSGSGSGTEFTLTISSL QHYDTYPYT FGQGTKLEIKR QPDDFAAYYC (SEQ ID(SEQ ID (SEQ ID NO:121) NO:122) NO:123) ING1 GVPSRFSGSGSGTDFTLTISSLQQSYSTPRTT FGGGTKVDIKR QPEDFATYYC (SEQ ID (SEQ ID (SEQ ID NO:124)NO:125) NO:126) ING2 GVPDRFSGSGSGTDFTLTISSL QQYYSTPFT FGGGTKVEIKRQAEDVAVYYC (SEQ ID (SEQ ID (SEQ ID NO:127) NO:128) NO:129) 2G11GVPSRFSGSRFGTDFTLTISSL QQSYSTRALT FGGGTKVEIKR QPEDFATYYC (SEQ ID (SEQ ID(SEQ ID NO:130) NO:131) NO:132) 5G4 GVPSRFSGSGSGTDFTLTISSL QQSYSTLMCSFGQGTKLEIKR QPEDVATYYC (SEQ ID (SEQ ID (SEQ ID NO:133) NO:134) NO:135)*Sequence for complete heavy chain is heavy chain framework 1 + CDR1 +framework 2 + CDR2 + framework 3 + CDR3 + framework 4. Sequence forcomplete light chain is light chain framework 1 + CDR1 + framework 2 +CDR2 + framework 3 + CDR3 + framework 4.

Using the teachings and the sequence information provided herein, thevariable light and variable heavy chains can be joined directly orthrough a linker (e.g. a (Gly₄Ser₃, SEQ ID NO:181) to form asingle-chain Fv antibody. The various CDRs and/or framework regions canbe used to form full human antibodies, chimeric antibodies, antibodyfragments, polyvalent antibodies, and the like.

III. Preparation of BoNT Neutralizing Antibodies.

A) Recombinant Expression of BoNT-Neutralizing Antibodies.

Using the information provided herein, the botulinumneurotoxin-neutralizing antibodies of this invention are prepared usingstandard techniques well known to those of skill in the art.

For example, the polypeptide sequences provided herein (see, e.g., Table2, and/or Table 6, and/or Table 9 and/or Table 13) can be used todetermine appropriate nucleic acid sequences encoding theBoNT/A-neutralizing antibodies and the nucleic acids sequences then usedto express one or more BoNT-neutralizing antibodies. The nucleic acidsequence may be optimized to reflect particular codon “preferences” forvarious expression systems according to standard methods well known tothose of skill in the art.

Using the sequence information provided, the nucleic acids may besynthesized according to a number of standard methods known to those ofskill in the art. Oligonucleotide synthesis, is preferably carried outon commercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) ormanually synthesized using the solid phase phosphoramidite triestermethod described by Beaucage et. al. (Beaucage et. al. (1981)Tetrahedron Letts. 22(20): 1859-1862).

Once a nucleic acid encoding a BoNT/A-neutralizing antibody issynthesized it may be amplified and/or cloned according to standardmethods. Molecular cloning techniques to achieve these ends are known inthe art. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant nucleic acids are known topersons of skill. Examples of these techniques and instructionssufficient to direct persons of skill through many cloning exercises arefound in Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual(2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY, (Sambrook); and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement) (Ausubel). Methods of producing recombinant immunoglobulinsare also known in the art. See, Cabilly, U.S. Pat. No. 4,816,567; andQueen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Qβ-replicase amplification andother RNA polymerase mediated techniques are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080;Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene4, 560; and Barringer et al. (1990) Gene 89, 117. Improved methods ofcloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039.

Once the nucleic acid for a BoNT/A-neutralizing antibody is isolated andcloned, one may express the gene in a variety of recombinantlyengineered cells known to those of skill in the art. Examples of suchcells include bacteria, yeast, filamentous fungi, insect (especiallyemploying baculoviral vectors), and mammalian cells. It is expected thatthose of skill in the art are knowledgeable in the numerous expressionsystems available for expression of BoNT/A-neutralizing antibodies.

In brief summary, the expression of natural or synthetic nucleic acidsencoding BoNT/A-neutralizing antibodies will typically be achieved byoperably linking a nucleic acid encoding the antibody to a promoter(which is either constitutive or inducible), and incorporating theconstruct into an expression vector. The vectors can be suitable forreplication and integration in prokaryotes, eukaryotes, or both. Typicalcloning vectors contain transcription and translation terminators,initiation sequences, and promoters useful for regulation of theexpression of the nucleic acid encoding the BoNT/A-neutralizingantibody. The vectors optionally comprise generic expression cassettescontaining at least one independent terminator sequence, sequencespermitting replication of the cassette in both eukaryotes andprokaryotes, i.e., shuttle vectors, and selection markers for bothprokaryotic and eukaryotic systems. See Sambrook.

To obtain high levels of expression of a cloned nucleic acid it iscommon to construct expression plasmids which typically contain a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.Examples of regulatory regions suitable for this purpose in E. coli arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky (1984) J. Bacteriol., 158:1018-1024 andthe leftward promoter of phage lambda (P_(L)) as described by Herskowitzand Hagen (1980) Ann. Rev. Genet., 14:399-445. The inclusion ofselection markers in DNA vectors transformed in E. coli is also useful.Examples of such markers include genes specifying resistance toampicillin, tetracycline, or chloramphenicol. See Sambrook for detailsconcerning selection markers, e.g., for use in E. coli.

Expression systems for expressing BoNT/A-neutralizing antibodies areavailable using E. coli, Bacillus sp. (Palva, et al. (1983) Gene22:229-235; Mosbach et al., Nature, 302: 543-545 and Salmonella. E. colisystems are preferred.

The BoNT/A-neutralizing antibodies produced by prokaryotic cells mayrequire exposure to chaotropic agents for proper folding. Duringpurification from, e.g., E. coli, the expressed protein is optionallydenatured and then renatured. This is accomplished, e.g., bysolubilizing the bacterially produced antibodies in a chaotropic agentsuch as guanidine HCl. The antibody is then renatured, either by slowdialysis or by gel filtration. See, U.S. Pat. No. 4,511,503.

Methods of transfecting and expressing genes in mammalian cells areknown in the art. Transducing cells with nucleic acids can involve, forexample, incubating viral vectors containing BoNT/A-neutralizing nucleicacids with cells within the host range of the vector. See, e.g., Goeddel(1990) Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego,Calif. or Krieger (1990) Gene Transfer and Expression—A LaboratoryManual, Stockton Press, New York, N.Y. and the references cited therein.

The culture of cells used in the present invention, including cell linesand cultured cells from tissue or blood samples is well known in the art(see, e.g., Freshney (1994) Culture of Animal Cells, a Manual of BasicTechnique, third edition, Wiley-Liss, N.Y. and the references citedtherein).

Techniques for using and manipulating antibodies are found in Coligan(1991) Current Protocols in Immunology Wiley/Greene, N.Y.; Harlow andLane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature256: 495-497. BoNT/A-neutralizing antibodies that are specific forbotulinum neurotoxin type A have a K_(D) of 1×10⁻⁸ M or better, withpreferred embodiments having a K_(D) of 1 nM or better and mostpreferred embodiments having a K_(D) of 0.1 nM or better.

In one preferred embodiment the BoNT/A-neutralizing antibody gene (e.g.BoNT/A-neutralizing scFv gene) is subcloned into the expression vectorpUC119mycHis (Tomlinson et al. (1996) J. Mol. Biol., 256: 813-817) orpSYN3, resulting in the addition of a hexahistidine tag at theC-terminal end of the scFv to facilitate purification. Detailedprotocols for the cloning and purification of BoNT/A-neutralizingantibodies are provided in Example 1.

B) Preparation of Whole Polyclonal or Monoclonal Antibodies.

The BoNT neutralizing antibodies of this invention include individual,allelic, strain, or species variants, and fragments thereof, both intheir naturally occurring (full-length) forms and in recombinant forms.In certain embodiments, preferred antibodies are selected to bind one ormore epitopes bound by the antibodies described herein (e.g., S25, C25,C39, 1C6, 3D12, B4, 1F3, HuC25, AR1, AR2, WR1(V), WR1(T), 3-1, 3-8,3-10, CR1, RAZ1, 1D11, 2G11, 5G4, ING1, and/or ING2). Certain preferredantibodies are cross-reactive with two or more BoNT subtypes (e.g.BoNT/A1, BoNT/A2, BoNT/A3, etc.). The antibodies can be raised in theirnative configurations or in non-native configurations. Anti-idiotypicantibodies can also be generated. Many methods of making antibodies thatspecifically bind to a particular epitope are known to persons of skill.The following discussion is presented as a general overview of thetechniques available; however, one of skill will recognize that manyvariations upon the following methods are known.

1) Polyclonal Antibody Production.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen (e.g., BoNT/A1 or A2, BoNT/A1 or A2H_(c), or BoNT/A1 or A2 subsequences including, but not limited tosubsequences comprising epitopes specifically bound by antibodiesexpressed by clones clones S25, C25, C39, 1C6, 3D12, B4, 1F3, HuC25,AR1, AR2, WR1(V), WR1(T), 3-1, 3-8, 3-10, and/or CR1, RAZ1, 1D11, 2G11,5G4, ING1, and/or ING2 disclosed herein), preferably a purifiedpolypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST,keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the polypeptide of interest. When appropriately hightiters of antibody to the immunogen are obtained, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the BoNT/A polypeptide isperformed where desired (see, e.g., Coligan (1991) Current Protocols inImmunology Wiley/Greene, N.Y.; and Harlow and Lane (1989) Antibodies: ALaboratory Manual Cold Spring Harbor Press, N.Y.).

Antibodies that specifically bind to the neutralizing epitopes describedherein can be selected from polyclonal sera using the selectiontechniques described herein.

2) Monoclonal Antibody Production.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Descriptions of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

Summarized briefly, monoclonal antibody production proceeds by injectingan animal with an immunogen (e.g., BoNT/A, BoNT/A H_(c), or BoNT/Asubsequences including, but not limited to subsequences comprisingepitopes specifically bound by antibodies expressed by clones S25, C25,C39, 1C6, 3D12, B4, 1F3, HuC25, AR1, AR2, WR1(V), WR1(T), 3-1, 3-8,3-10, and/or CR1, RAZ1, 1D11, 2G11, 5G4, ING1, and/or ING2 disclosedherein). The animal is then sacrificed and cells taken from its spleen,which are fused with myeloma cells. The result is a hybrid cell or“hybridoma” that is capable of reproducing in vitro. The population ofhybridomas is then screened to isolate individual clones, each of whichsecrete a single antibody species to the immunogen. In this manner, theindividual antibody species obtained are the products of immortalizedand cloned single B cells from the immune animal generated in responseto a specific site recognized on the immunogenic substance.

Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe BoNT antigen, and yield of the monoclonal antibodies produced bysuch cells is enhanced by various techniques, including injection intothe peritoneal cavity of a vertebrate (preferably mammalian) host. Theantibodies of the present invention are used with or withoutmodification, and include chimeric antibodies such as humanized murineantibodies.

IV. Modification of BoNT Neutralizing Antibodies.

A) Phage Display Can Be Used to Increase Antibody Affinity.

To create higher affinity antibodies, mutant scFv gene repertories,based on the sequence of a binding scFv (e.g., Table 2, and/or Table 6,and/or Table 9 and/or Table 13), can be created and expressed on thesurface of phage. Display of antibody fragments on the surface ofviruses which infect bacteria (bacteriophage or phage) makes it possibleto produce human or other mammalian antibodies (e.g. scFvs) with a widerange of affinities and kinetic characteristics. To display antibodyfragments on the surface of phage (phage display), an antibody fragmentgene is inserted into the gene encoding a phage surface protein (e.g.,pIII) and the antibody fragment-pIII fusion protein is expressed on thephage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboomet al. (1991) Nucleic Acids Res., 19: 4133-4137).

Since the antibody fragments on the surface of the phage are functional,those phage bearing antigen binding antibody fragments can be separatedfrom non-binding or lower affinity phage by antigen affinitychromatography (McCafferty et al. (1990) Nature, 348: 552-554). Mixturesof phage are allowed to bind to the affinity matrix, non-binding orlower affinity phage are removed by washing, and bound phage are elutedby treatment with acid or alkali. Depending on the affinity of theantibody fragment, enrichment factors of 20 fold-1,000,000 fold areobtained by single round of affinity selection.

By infecting bacteria with the eluted phage or modified variants of theeluted phage as described below, more phage can be grown and subjectedto another round of selection. In this way, an enrichment of 1000 foldin one round becomes 1,000,000 fold in two rounds of selection(McCafferty et al. (1990) Nature, 348: 552-554). Thus, even whenenrichments in each round are low, multiple rounds of affinity selectionleads to the isolation of rare phage and the genetic material containedwithin which encodes the sequence of the binding antibody (Marks et al.(1991) J. Mol. Biol., 222: 581-597). The physical link between genotypeand phenotype provided by phage display makes it possible to test everymember of an antibody fragment library for binding to antigen, even withlibraries as large as 100,000,000 clones. For example, after multiplerounds of selection on antigen, a binding scFv that occurred with afrequency of only 1/30,000,000 clones was recovered (Id.).

1) Chain Shuffling.

One approach for creating mutant scFv gene repertoires involvesreplacing either the V_(H) or V_(L) gene from a binding scFv with arepertoire of V_(H) or V_(L) genes (chain shuffling) (Clackson et al.(1991) Nature, 352: 624-628). Such gene repertoires contain numerousvariable genes derived from the same germline gene as the binding scFv,but with point mutations (Marks et al. (1992) Bio/Technology, 10:779-783). Using light or heavy chain shuffling and phage display, thebinding avidities of, e.g., BoNT/A1/BoNT/A2-neutralizing antibodyfragment can be dramatically increased (see, e.g., Marks et al. (1992)Bio/Technology, 10: 779-785 in which the affinity of a human scFvantibody fragment which bound the hapten phenyloxazolone (phox) wasincreased from 300 nM to 15 nM (20 fold)).

Thus, to alter the affinity of BoNT-neutralizing antibody a mutant scFvgene repertoire is created containing the V_(H) gene of a knownBoNT-neutralizing antibody (e.g., CR1, RAZ1, ING1, ING2) and a V_(L)gene repertoire (light chain shuffling). Alternatively, an scFv generepertoire is created containing the V_(L) gene of a knownBoNT-neutralizing antibody (e.g., CR1, RAZ1, ING1, ING2) and a V_(H)gene repertoire (heavy chain shuffling). The scFv gene repertoire iscloned into a phage display vector (e.g., pHEN-1, Hoogenboom et al.(1991) Nucleic Acids Res., 19: 4133-4137) and after transformation alibrary of transformants is obtained. Phage were prepared andconcentrated and selections are performed as described in the examples.

The antigen concentration is decreased in each round of selection,reaching a concentration less than the desired K_(d) by the final roundsof selection. This results in the selection of phage on the basis ofaffinity (Hawkins et al. (1992) J. Mol. Biol. 226: 889-896).

2) Increasing the Affinity of BoNT-Neutralizing Antibodies by SiteDirected Mutagenesis.

The majority of antigen contacting amino acid side chains are located inthe complementarity determining regions (CDRs), three in the V_(H)(CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, and CDR3)(Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothia et al.(1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol., 217:133-151). These residues contribute the majority of binding energeticsresponsible for antibody affinity for antigen. In other molecules,mutating amino acids that contact ligand has been shown to be aneffective means of increasing the affinity of one protein molecule forits binding partner (Lowman et al. (1993) J. Mol. Biol., 234: 564-578;Wells (1990) Biochemistry, 29: 8509-8516). Thus mutation (randomization)of the CDRs and screening against BoNT/A, BoNT/A H_(C) or the epitopesthereof identified herein, may be used to generate BoNT/A-neutralizingantibodies having improved binding affinity.

In certain embodiments, each CDR is randomized in a separate library,using, for example, S25 as a template (K_(d)=7.3×10⁻⁸ M). To simplifyaffinity measurement, S25, or other lower affinity BoNT/A-neutralizingantibodies, are used as a template, rather than a higher affinity scFv.The CDR sequences of the highest affinity mutants from each CDR libraryare combined to obtain an additive increase in affinity. A similarapproach has been used to increase the affinity of human growth hormone(hGH) for the growth hormone receptor over 1500 fold from 3.4×10⁻¹⁰ to9.0×10⁻¹³ M (Lowman et al. (1993) J. Mol. Biol., 234: 564-578).

To increase the affinity of BoNT-neutralizing antibodies, amino acidresidues located in one or more CDRs (e.g., 9 amino acid residueslocated in V_(L) CDR3) are partially randomized by synthesizing a“doped” oligonucleotide in which the wild type nucleotide occurred witha frequency of, e.g. 49%. The oligonucleotide is used to amplify theremainder of the BoNT-neutralizing scFv gene(s) using PCR.

For example in one embodiment, to create a library in which V_(H) CDR3is randomized an oligonucleotide is synthesized which anneals to theBoNT-neutralizing antibody V_(H) framework 3 and encodes V_(H) CDR3 anda portion of framework 4. At the four positions to be randomized, thesequence NNS can be used, where N is any of the 4 nucleotides, and S is“C” or “T”. The oligonucleotide is used to amplify theBoNT/A-neutralizing antibody V_(H) gene using PCR, creating a mutantBoNT-neutralizing antibody V_(H) gene repertoire. PCR is used to splicethe V_(H) gene repertoire with the BoNT-neutralizing antibody lightchain gene, and the resulting scFv gene repertoire cloned into a phagedisplay vector (e.g., pHEN-1 or pCANTAB5E). Ligated vector DNA is usedto transform electrocompetent E. coli to produce a phage antibodylibrary.

To select higher affinity mutant scFv, each round of selection of thephage antibody libraries is conducted on decreasing amounts of one ormore BoNT subtypes, as described in the Examples. Typically, 96 clonesfrom the third and fourth round of selection can screened for binding tothe desired antigen(s) (e.g., BoNT/A1 and/or BoNT/A2) by ELISA on 96well plates. scFv from, e.g., twenty to forty ELISA positive clones areexpressed, e.g. in 10 ml cultures, the periplasm harvested, and the scFvk_(off) determined by BIAcore. Clones with the slowest k_(off) aresequenced, and each unique scFv subcloned into an appropriate vector(e.g., pUC119 mycHis). The scFv are expressed in culture, and purifiedas described herein. Affinities of purified scFv are determined byBIAcore.

3) Creation of BoNT-Neutralizing (scFv′)2 Homodimers.

To create BoNT-neutralizing (scFv′)₂ antibodies, two BoNT-neutralizingscFvs are joined, either through a linker (e.g., a carbon linker, apeptide, etc.) or through a disulfide bond between, for example, twocysteins. Thus, for example, to create disulfide linkedBoNT/A-neutralizing scFv, a cysteine residue can be introduced by sitedirected mutagenesis between the myc tag and hexahistidine tag at thecarboxy-terminus of the BoNT/A-neutralizing scFv. Introduction of thecorrect sequence is verified by DNA sequencing. In a preferredembodiment, the construct is in pUC119, so that the pelB leader directsexpressed scFv to the periplasm and cloning sites (Ncol and Notl) existto introduce BoNT/A-neutralizing mutant scFv. Expressed scFv has the myctag at the C-terminus, followed by two glycines, a cysteine, and then 6histidines to facilitate purification by IMAC. After disulfide bondformation between the two cysteine residues, the two scFv are separatedfrom each other by 26 amino acids (two 11 amino acid myc tags and 4glycines). An scFv was expressed from this construct, purified by IMACmay predominantly comprise monomeric scFv. To produce (scFv′)₂ dimers,the cysteine is reduced by incubation with 1 MM beta-mercaptoethanol,and half of the scFv blocked by the addition of DTNB. Blocked andunblocked scFvs are incubated together to form (scFv′)₂ and theresulting material can optionally be analyzed by gel filtration. Theaffinity of the BoNT-neutralizing scFv′ monomer and (scFv′)₂ dimer canoptionally be determined by BIAcore as described herein.

In a particularly preferred embodiment, the (scFv′)₂ dimer is created byjoining the scFv fragments through a linker, more preferably through apeptide linker. This can be accomplished by a wide variety of means wellknown to those of skill in the art. For example, one preferred approachis described by Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448 (see also WO 94/13804).

Typically, linkers are introduced by PCR cloning. For example, syntheticoligonucleotides encoding the 5 amino acid linker (G₄S, SEQ ID NO:136)can be used to PCR amplify the BoNT/A-neutralizing antibody V_(H) andV_(L) genes which are then spliced together to create theBoNT/A-neutralizing diabody gene. The gene is then cloned into anappropriate vector, expressed, and purified according to standardmethods well known to those of skill in the art.

4) Preparation of BoNT-Neutralizing (scFv)₂, Fab, and (Fab′)₂ Molecules.

BoNT-neutralizing antibodies such as BoNT/A1-A2-neutralizing scFv, orvariant(s) with higher affinity, are suitable templates for creatingsize and valency variants. For example, a BoNT/A1-A2-neutralizing(scFv′)₂ can be created from the parent scFv (e.g. CR1, RAZ1, ING1,ING2, etc.) as described above. An scFv gene can be excised usingappropriate restriction enzymes and cloned into another vector asdescribed herein.

In one embodiment, expressed scFv has a myc tag at the C-terminus,followed by two glycines, a cysteine, and six histidines to facilitatepurification. After disulfide bond formation between the two cystineresidues, the two scFv should be separated from each other by 26 aminoacids (e.g., two eleven amino acid myc tags and four glycines). scFv isexpressed from this construct and purified.

To produce (scFv′)₂ dimers, the cysteine is reduced by incubation with 1mM β-mercaptoethanol, and half of the scFv blocked by the addition ofDTNB. Blocked and unblocked scFv are incubated together to form(scFv′)₂, which is purified. As higher affinity scFv are isolated, theirgenes are similarly used to construct (scFv′)₂.

BoNT/A-neutralizing Fab are expressed in E. coli using an expressionvector similar to the one described by Better et. al. (1988) Science,240: 1041-1043. To create a BoNT/A-neutralizing Fab, the V_(H) and V_(L)genes are amplified from the scFv using PCR. The V_(H) gene is clonedinto an expression vector (e.g., a PUC119 based bacterial expressionvector) that provides an IgG C_(H)1 domain downstream from, and in framewith, the V_(H) gene. The vector also contains the lac promoter, a pelbleader sequence to direct expressed V_(H)-C_(H)1 domain into theperiplasm, a gene 3 leader sequence to direct expressed light chain intothe periplasm, and cloning sites for the light chain gene. Clonescontaining the correct V_(H) gene are identified, e.g., by PCRfingerprinting. The V_(L) gene is spliced to the CL gene using PCR andcloned into the vector containing the V_(H) C_(H)1 gene.

B) Selection of Neutralizing Antibodies.

In preferred embodiments, selection of BoNT-neutralizing antibodies(whether produced by phage display, yeast display, immunization methods,hybridoma technology, etc.) involves screening the resulting antibodiesfor specific binding to an appropriate antigen(s). In the instant case,suitable antigens include, but are not limited to BoNT/A1, BoNT/A2,BoNT/A3 H_(C), a C-terminal domain of BoNT heavy chain (binding domain),BoNT/A3 holotoxins, or recombinant BoNT domains such as HC (bindingdomain), HN (translocation domain), or LC (light chain). In particularlypreferred embodiments the neutralizing antibodies are selected forspecific binding of an epitope recognized by one or more of theantibodies described herein.

Selection can be by any of a number of methods well known to those ofskill in the art. In a preferred embodiment, selection is byimmunochromatography (e.g., using immunotubes, Maxisorp, Nunc) againstthe desired target, e.g., BoNT/A or BoNT/A H_(C). In another embodiment,selection is against a BoNT HC in surface plasmon resonance system(e.g., BIAcore, Pharmacia) either alone or in combination with anantibody that binds to an epitope specifically bound by one or more ofthe antibodies described herein. Selection can also be done using flowcytometry for yeast display libraries. In one preferred embodiment,yeast display libraries are sequentially selected, first on BoNT/A1,then on BoNT/A2 to obtain antibodies that bind with high affinity toboth subtypes of BoNT/A. This can be repeated for other subtypes.

For phage display, analysis of binding can be simplified by including anamber codon between the antibody fragment gene and gene III. This makesit possible to easily switch between displayed and soluble antibodyfragments simply by changing the host bacterial strain. When phage aregrown in a supE suppresser strain of E. coli, the amber stop codonbetween the antibody gene and gene III is read as glutamine and theantibody fragment is displayed on the surface of the phage. When elutedphage are used to infect a non-suppressor strain, the amber codon isread as a stop codon and soluble antibody is secreted from the bacteriainto the periplasm and culture media (Hoogenboom et al. (1991) NucleicAcids Res., 19: 4133-4137). Binding of soluble scFv to antigen can bedetected, e.g., by ELISA using a murine IgG monoclonal antibody (e.g.,9E1O) which recognizes a C-terminal myc peptide tag on the scFv (Evan etal. (1985) Mol. Cell Biol., 5: 3610-3616; Munro et al. (1986) Cell, 46:291-300), e.g., followed by incubation with polyclonal anti-mouse Fcconjugated to a detectable label (e.g., horseradish peroxidase).

As indicated above, purification of the BoNT-neutralizing antibody canbe facilitated by cloning of the scFv gene into an expression vector(e.g., expression vector pUC119mycHIS) that results in the addition ofthe myc peptide tag followed by a hexahistidine tag at the C-terminalend of the scFv. The vector also preferably encodes the pectate lyaseleader sequence that directs expression of the scFv into the bacterialperiplasm where the leader sequence is cleaved. This makes it possibleto harvest native properly folded scFv directly from the bacterialperiplasm. The BoNT-neutralizing antibody is then expressed and purifiedfrom the bacterial supernatant using immobilized metal affinitychromatography.

C) Measurement of BoNT-Neutralizing Antibody Affinity for One or MoreBoNT Subtypes.

As explained above, selection for increased avidity involves measuringthe affinity of a BoNT-neutralizing antibody (or a modifiedBoNT-neutralizing antibody) for one or more targets of interest (e.g.BoNT/A subtype(s) or domains thereof, e.g. Hc or other epitope). Methodsof making such measurements are described in detail in the examplesprovided herein. Briefly, for example, the K_(d) of aBoNT/A-neutralizing antibody and the kinetics of binding to BoNT/A aredetermined in a BIAcore, a biosensor based on surface plasmon resonance.For this technique, antigen is coupled to a derivatized sensor chipcapable of detecting changes in mass. When antibody is passed over thesensor chip, antibody binds to the antigen resulting in an increase inmass that is quantifiable. Measurement of the rate of association as afunction of antibody concentration can be used to calculate theassociation rate constant (k_(on)). After the association phase, bufferis passed over the chip and the rate of dissociation of antibody(k_(off)) determined. K_(on) is typically measured in the range 1.0×10²to 5.0×10⁶ and k_(off) in the range 1.0×10⁻¹ to 1.0×10⁻⁶. Theequilibrium constant K_(d) is then calculated as k_(off)/k_(on) and thusis typically measured in the range 10⁻⁵ to 10⁻¹². Affinities measured inthis manner correlate well with affinities measured in solution byfluorescence quench titration.

Phage display and selection generally results in the selection of higheraffinity mutant scFvs (Marks et al. (1992) Bio/Technology, 10: 779-783;Hawkins et al. (1992) J. Mol. Biol. 226: 889-896; Riechmann et al.(1993) Biochemistry, 32: 8848-8855; Clackson et al. (1991) Nature, 352:624-628), but probably does not result in the separation of mutants withless than a 6 fold difference in affinity (Riechmann et al. (1993)Biochemistry, 32: 8848-8855). Thus a rapid method is needed to estimatethe relative affinities of mutant scFvs isolated after selection. Sinceincreased affinity results primarily from a reduction in the k_(off),measurement of k_(off) should identify higher affinity scFv. k_(off) canbe measured in the BIAcore on unpurified scFv in bacterial periplasm,since expression levels are high enough to give an adequate bindingsignal and k_(off) is independent of concentration. The value of k_(off)for periplasmic and purified scFv is typically in close agreement.

V. Human or Humanized (Chimeric) Antibody Production.

As indicated above, the BoNT-neutralizing antibodies of this inventioncan be administered to an organism (e.g., a human patient) fortherapeutic purposes (e.g., the treatment of botulism). Antibodiesadministered to an organism other than the species in which they areraised can be immunogenic. Thus, for example, murine antibodiesrepeatedly administered to a human often induce an immunologic responseagainst the antibody (e.g., the human anti-mouse antibody (HAMA)response). While this is typically not a problem for the use ofnon-human antibodies of this invention as they are typically notutilized repeatedly, the immunogenic properties of the antibody arereduced by altering portions, or all, of the antibody intocharacteristically human sequences thereby producing chimeric or humanantibodies, respectively.

A) Chimeric Antibodies.

Chimeric) antibodies are immunoglobulin molecules comprising a human andnon-human portion. More specifically, the antigen combining region (orvariable region) of a chimeric antibody is derived from a non-humansource (e.g., murine) and the constant region of the chimeric antibody(which confers biological effector function to the immunoglobulin) isderived from a human source. The chimeric antibody should have theantigen binding specificity of the non-human antibody molecule and theeffector function conferred by the human antibody molecule. A largenumber of methods of generating chimeric antibodies are well known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167,5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and4,975,369).

In general, the procedures used to produce chimeric antibodies consistof the following steps (the order of some steps may be interchanged):(a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains (or simply asthe V or variable region) may be in either the cDNA or genomic form; (b)cloning the gene segments encoding the constant region or desired partthereof; (c) ligating the variable region to the constant region so thatthe complete chimeric antibody is encoded in a transcribable andtranslatable form; (d) ligating this construct into a vector containinga selectable marker and gene control regions such as promoters,enhancers and poly(A) addition signals; (e) amplifying this construct ina host cell (e.g., bacteria); (f) introducing the DNA into eukaryoticcells (transfection) most often mammalian lymphocytes; and culturing thehost cell under conditions suitable for expression of the chimericantibody.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al. (1984) Nature, 312: 643; and anti-tumorantigens: Sahagan et al. (1986) J. Immunol., 137: 1066). Likewiseseveral different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes (Neuberger et al. (1984) Nature 312: 604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al. (1984) Nature309: 364; Tan et al., (1985) J. Immunol. 135: 3565-3567).

In one preferred embodiment, a recombinant DNA vector is used totransfect a cell line that produces a BoNT/A-neutralizing antibody. Thenovel recombinant DNA vector contains a “replacement gene” to replaceall or a portion of the gene encoding the immunoglobulin constant regionin the cell line (e.g., a replacement gene may encode all or a portionof a constant region of a human immunoglobulin, a specificimmunoglobulin class, or an enzyme, a toxin, a biologically activepeptide, a growth factor, inhibitor, or a linker peptide to facilitateconjugation to a drug, toxin, or other molecule, etc.), and a “targetsequence” which allows for targeted homologous recombination withimmunoglobulin sequences within the antibody producing cell.

In another embodiment, a recombinant DNA vector is used to transfect acell line that produces an antibody having a desired effector function,(e.g., a constant region of a human immunoglobulin) in which case, thereplacement gene contained in the recombinant vector may encode all or aportion of a region of an BoNT/A-neutralizing antibody and the targetsequence contained in the recombinant vector allows for homologousrecombination and targeted gene modification within the antibodyproducing cell. In either embodiment, when only a portion of thevariable or constant region is replaced, the resulting chimeric antibodymay define the same antigen and/or have the same effector function yetbe altered or improved so that the chimeric antibody may demonstrate agreater antigen specificity, greater affinity binding constant,increased effector function, or increased secretion and production bythe transfected antibody producing cell line, etc.

Regardless of the embodiment practiced, the processes of selection forintegrated DNA (via a selectable marker), screening for chimericantibody production, and cell cloning, can be used to obtain a clone ofcells producing the chimeric antibody.

Thus, a piece of DNA which encodes a modification for a monoclonalantibody can be targeted directly to the site of the expressedimmunoglobulin gene within a B-cell or hybridoma cell line. DNAconstructs for any particular modification may be used to alter theprotein product of any monoclonal cell line or hybridoma. Such aprocedure circumvents the costly and time consuming task of cloning bothheavy and light chain variable region genes from each B-cell cloneexpressing a useful antigen specificity. In addition to circumventingthe process of cloning variable region genes, the level of expression ofchimeric antibody should be higher when the gene is at its naturalchromosomal location rather than at a random position. Detailed methodsfor preparation of chimeric (humanized) antibodies can be found in U.S.Pat. No. 5,482,856.

B) Human and Humanized Antibodies.

In another embodiment, this invention provides for humanized or fullyhuman anti-BoNT-neutralizing antibodies (e.g. HuC25, RAZ1, CR1, ING1,ING2, etc.). Human antibodies consist entirely of characteristicallyhuman polypeptide sequences. The human BoNT-neutralizing antibodies ofthis invention can be produced in using a wide variety of methods (see,e.g., Larrick et al., U.S. Pat. No. 5,001,065, for review).

In certain preferred embodiments, fully human scFv antibodies of thisinvention are obtained by modification and screening of fully humansingle-chain (e.g. scFv) libraries. Thus, in certain embodiments, fullyhuman antibodies are produced using phage and/or yeast display methodsas described herein. Methods of producing fully human gene libraries arewell known to those of skill in the art (see, e.g., Vaughn et al. (1996)Nature Biotechnology, 14(3): 309-314, Marks et al. (1991) J. Mol. Biol.,222: 581-597, and PCT/US96/10287).

In another embodiment, human BoNT-neutralizing antibodies of the presentinvention are can be produced in trioma cells. Genes encoding theantibodies are then cloned and expressed in other cells, particularly,nonhuman mammalian cells.

The general approach for producing human antibodies by trioma technologyhas been described by Ostberg et al. (1983) Hybridoma 2: 361-367,Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.4,634,666. The antibody-producing cell lines obtained by this method arecalled triomas because they are descended from three cells; two humanand one mouse. Triomas have been found to produce antibody more stablythan ordinary hybridomas made from human cells.

Preparation of trioma cells requires an initial fusion of a mousemyeloma cell line with unimmunized human peripheral B lymphocytes. Thisfusion generates a xenogenic hybrid cell containing both human and mousechromosomes (see, Engelman, supra.). Xenogenic cells that have lost thecapacity to secrete antibodies are selected. Preferably, a xenogeniccell is selected that is resistant to 8-azaguanine. Such cells areunable to propagate on hypoxanthine-aminopterin-thymidine (HAT) orazaserine-hypoxanthine (AH) media.

The capacity to secrete antibodies is conferred by a further fusionbetween the xenogenic cell and B-lymphocytes immunized against a BoNTpolypeptide (e.g., BoNT/A, BoNT/A H_(c), BoNT/A subsequences including,but not limited to subsequences comprising epitopes specifically boundby the antibodies described herein, etc.). The B-lymphocytes areobtained from the spleen, blood or lymph nodes of human donor. Ifantibodies against a specific antigen or epitope are desired, it ispreferable to use that antigen or epitope thereof as the immunogenrather than the entire polypeptide. Alternatively, B-lymphocytes areobtained from an unimmunized individual and stimulated with a BoNTpolypeptide, or a epitope thereof, in vitro. In a further variation,B-lymphocytes are obtained from an infected, or otherwise immunizedindividual, and then hyperimmunized by exposure to a BoNT polypeptidefor about seven to fourteen days, in vitro.

The immunized B-lymphocytes prepared by one of the above procedures arefused with a xenogenic hybrid cell by well known methods. For example,the cells are treated with 40-50% polyethylene glycol of MW 1000-4000,at about 37° C. for about 5-10 min. Cells are separated from the fusionmixture and propagated in media selective for the desired hybrids. Whenthe xenogenic hybrid cell is resistant to 8-azaguanine, immortalizedtrioma cells are conveniently selected by successive passage of cells onHAT or AH medium. Other selective procedures are, of course, possibledepending on the nature of the cells used in fusion. Clones secretingantibodies having the required binding specificity are identified byassaying the trioma culture medium for the ability to bind to the BoNTpolypeptide or an epitope thereof. Triomas producing human antibodieshaving the desired specificity are subcloned by the limiting dilutiontechnique and grown in vitro in culture medium, or are injected intoselected host animals and grown in vivo.

The trioma cell lines obtained are then tested for the ability to bind aBoNT polypeptide or an epitope thereof. Antibodies are separated fromthe resulting culture medium or body fluids by conventionalantibody-fractionation procedures, such as ammonium sulfateprecipitation, DEAE cellulose chromatography and affinitychromatography.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into a cell line such as the cell linestypically used for expression of recombinant or humanizedimmunoglobulins. As well as increasing yield of antibody, this strategyoffers the additional advantage that immunoglobulins are obtained from acell line that does not have a human component, and does not thereforeneed to be subjected to the especially extensive viral screeningrequired for human cell lines.

The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingbut not limited to, the polymerase chain reaction (PCR), known in theart (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor, N.Y., 1989; Berger & Kimmel, Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, AcademicPress, Inc., San Diego, Calif., 1987; Co et al. (1992) J. Immunol., 148:1149). For example, genes encoding heavy and light chains are clonedfrom a trioma's genomic DNA or cDNA produced by reverse transcription ofthe trioma's RNA. Cloning is accomplished by conventional techniquesincluding the use of PCR primers that hybridize to the sequencesflanking or overlapping the genes, or segments of genes, to be cloned.

Typically, recombinant constructs comprise DNA segments encoding acomplete human immunoglobulin heavy chain and/or a complete humanimmunoglobulin light chain of an immunoglobulin expressed by a triomacell line. Alternatively, DNA segments encoding only a portion of theprimary antibody genes are produced, which portions possess bindingand/or effector activities. Other recombinant constructs containsegments of trioma cell line immunoglobulin genes fused to segments ofother immunoglobulin genes, particularly segments of other humanconstant region sequences (heavy and/or light chain). Human constantregion sequences can be selected from various reference sources,including but not limited to those listed in Kabat et al. (1987)Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services.

In addition to the DNA segments encoding BoNT/A-neutralizingimmunoglobulins or fragments thereof, other substantially homologousmodified immunoglobulins can be readily designed and manufacturedutilizing various recombinant DNA techniques known to those skilled inthe art such as site-directed mutagenesis (see Gillman & Smith (1979)Gene, 8: 81-97; Roberts et al. (1987) Nature 328: 731-734). Suchmodified segments will usually retain antigen binding capacity and/oreffector function. Moreover, the modified segments are usually not sofar changed from the original trioma genomic sequences to preventhybridization to these sequences under stringent conditions. Because,like many genes, immunoglobulin genes contain separate functionalregions, each having one or more distinct biological activities, thegenes may be fused to functional regions from other genes to producefusion proteins (e.g., immunotoxins) having novel properties or novelcombinations of properties.

The genomic sequences can be cloned and expressed according to standardmethods as described herein.

Other approaches to antibody production include in vitro immunization ofhuman blood. In this approach, human blood lymphocytes capable ofproducing human antibodies are produced. Human peripheral blood iscollected from the patient and is treated to recover mononuclear cells.The suppressor T-cells then are removed and remaining cells aresuspended in a tissue culture medium to which is added the antigen andautologous serum and, preferably, a nonspecific lymphocyte activator.The cells then are incubated for a period of time so that they producethe specific antibody desired. The cells then can be fused to humanmyeloma cells to immortalize the cell line, thereby to permit continuousproduction of antibody (see U.S. Pat. No. 4,716,111).

In another approach, mouse-human hybridomas which produce humanBoNT-neutralizing antibodies are prepared (see, e.g., U.S. Pat. No.5,506,132). Other approaches include immunization of murines transformedto express human immunoglobulin genes, and phage display screening(Vaughan et al. supra.).

VI. Assaying for Cross-Reactivity at a Neutralizing Epitope.

In a preferred embodiment, the antibodies of this invention specificallybind to one or more epitopes recognized by antibodies described herein(e.g. S25, C25, C39, 1C6, 1F3, CR1, 3D12, RAZ1, ING1, ING2, etc.). Inother words, particularly preferred antibodies are cross-reactive withone of more of these antibodies. Means of assaying for cross-reactivityare well known to those of skill in the art (see, e.g., Dowbenko et al.(1988) J. Virol. 62: 4703-4711).

This can be ascertained by providing one or more isolated target BoNTpolypeptide(s) (e.g. BoNT/A1 and/or BoNT/A2, or recombinant domains ofsaid toxin, such as H_(c)) attached to a solid support and assaying theability of a test antibody to compete with, e.g., S25, C25, C39, 1C6,1F3, CR1, 3D12, RAZ1, ING1, and/or ING2, etc for binding to the targetBoNT peptide. Thus, immunoassays in a competitive binding format arepreferably used for crossreactivity determinations. For example, in oneembodiment, a BoNT/A1 and/or A2 H_(C) polypeptide is immobilized to asolid support. Antibodies to be tested (e.g. generated by selection froma phage-display library) added to the assay compete with S25, C25, C39,1C6, 1F3, CR1, 3D12, RAZ1, ING1, ING2, etc antibodies binding to theimmobilized BoNT polypeptide(s). The ability of test antibodies tocompete with the binding of the S25, C25, C39, 1C6, 1F3, CR1, 3D12,RAZ1, ING1, and/or ING2, etc antibodies to the immobilized protein arecompared. The percent crossreactivity above proteins is then calculated,using standard calculations.

If the test antibody competes with one or more of the S25, C25, C39,1C6, 1F3, CR1, 3D12, RAZ1, ING1, and/or ING2, etc antibodies and has abinding affinity comparable to or greater than about 1×10⁻⁸ M with thesame target then the test antibody is expected to be a BoNT-neutralizingantibody.

In a particularly preferred embodiment, cross-reactivity is performed byusing surface plasmon resonance in a BIAcore. In a BIAcore flow cell,the BoNT polypeptide(s) (e.g., BoNT/A1 and/or BoNT/A2 H_(c)) are coupledto a sensor chip (e.g. CM5) as described in the examples. With a flowrate of 5 μl/min, a titration of 100 nM to 1 μM antibody is injectedover the flow cell surface for about 5 minutes to determine an antibodyconcentration that results in near saturation of the surface. Epitopemapping or cross-reactivity is then evaluated using pairs of antibodiesat concentrations resulting in near saturation and at least 100 RU ofantibody bound. The amount of antibody bound is determined for eachmember of a pair, and then the two antibodies are mixed together to givea final concentration equal to the concentration used for measurementsof the individual antibodies. Antibodies recognizing different epitopesshow an essentially additive increase in the RU bound when injectedtogether, while antibodies recognizing identical epitopes show only aminimal increase in RU (see the examples). In a particularly preferredembodiment, antibodies are said to be cross-reactive if, when “injected”together they show an essentially additive increase (preferably anincrease by at least a factor of about 1.4, more preferably an increaseby at least a factor of about 1.6, and most preferably an increase by atleast a factor of about 1.8 or 2.

Cross-reactivity at the desired epitopes can ascertained by a number ofother standard techniques (see, e.g., Geysen et al (1987) J. Immunol.Meth. 102, 259-274). This technique involves the synthesis of largenumbers of overlapping BoNT peptides. The synthesized peptides are thenscreened against one or more of the prototypical antibodies (e.g., CR1,RAZ1, ING1, ING2, etc.) and the characteristic epitopes specificallybound by these antibodies can be identified by binding specificity andaffinity. The epitopes thus identified can be conveniently used forcompetitive assays as described herein to identify cross-reactingantibodies.

The peptides for epitope mapping can be conveniently prepared using“Multipin” peptide synthesis techniques (see, e.g., Geysen et al (1987)Science, 235: 1184-1190). Using the known sequence of one or more BoNTsubtypes (see, e.g., Atassi et al. (1996) J. Prot. Chem., 7: 691-700 andreferences cited therein), overlapping BoNT polypeptide sequences can besynthesized individually in a sequential manner on plastic pins in anarray of one or more 96-well microtest plate(s).

The procedure for epitope mapping using this multipin peptide system isdescribed in U.S. Pat. No. 5,739,306. Briefly, the pins are firsttreated with a pre-coat buffer containing 2% bovine serum albumin and0.1% Tween 20 in PBS for 1 hour at room temperature. Then the pins arethen inserted into the individual wells of 96-well microtest platecontaining the antibodies in the pre-coat buffer, e.g. at 2 μg/ml. Theincubation is preferably for about 1 hour at room temperature. The pinsare washed in PBST (e.g., 3 rinses for every 10 minutes), and thenincubated in the wells of a 96-well microtest plate containing 100 mu 1of HRP-conjugated goat anti-mouse IgG (Fc) (Jackson ImmunoResearchLaboratories) at a 1:4,000 dilution for 1 hour at room temperature.After the pins are washed as before, the pins are put into wellscontaining peroxidase substrate solution of diammonium2,2′-azino-bis[3-ethylbenzthiazoline-b-sulfonate] (ABTS) and H₂O₂(Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) for 30 minutesat room temperature for color reaction. The plate is read at 405 nm by aplate reader (e.g., BioTek ELISA plate reader) against a backgroundabsorption wavelength of 492 nm. Wells showing color developmentindicated reactivity of the BoNT/A H_(C) peptides in such wells withS25, C25, C39, 1C6, or 1F3 antibodies.

VII. Assaying for Neutralizing Activity of Anti-BoNT Antibodies.

Preferred antibodies of this invention act, individually or incombination, to neutralize (reduce or eliminate) the toxicity ofbotulinum neurotoxin type A. Neutralization can be evaluated in vivo orin vitro. In vivo neutralization measurements simply involve measuringchanges in the lethality (e.g., LD₅₀ or other standard metric) due to aBoNT neurotoxin administration due to the presence of one or moreantibodies being tested for neutralizing activity. The neurotoxin can bedirectly administered to the test organism (e.g. mouse) or the organismcan harbor a botulism infection (e.g., be infected with Clostridiumbotulinum). The antibody can be administered before, during, or afterthe injection of BoNT neurotoxin or infection of the test animal. Adecrease in the rate of progression, or mortality rate indicates thatthe antibody(s) have neutralizing activity.

One suitable in vitro assay for neutralizing activity uses ahemidiaphragm preparation (Deshpande et al. (1995) Toxicon, 33:551-557). Briefly, left and right phrenic nerve hemidiaphragmpreparations are suspended in physiological solution and maintained at aconstant temperature (e.g. 36° C.). The phrenic nerves are stimulatedsupramaximally (e.g. at 0.05 Hz with square waves of 0.2 ms duration).Isometric twitch tension is measured with a force displacementtransducer (e.g., GrassModel FT03) connected to a chart recorder.

Purified antibodies are incubated with purified BoNT (e.g. BoNT/A1,BoNT/A2, BoNT/B, etc.) for 30 min at room temperature and then added tothe tissue bath, resulting in a final antibody concentration of about2.0×10⁻⁸ M and a final BoNT concentration of about 2.0×10⁻¹¹ M. For eachantibody studied, time to 50% twitch tension reduction is determined(e.g., three times for BoNT alone and three times for antibody plusBoNT). Differences between times to a given (arbitrary) percentage (e.g.50%) twitch reduction are determined by standard statistical analyses(e.g. two-tailed t test) at standard levels of significance (e.g., a Pvalue of <0.05 considered significant).

VIII. Diagnostic Assays.

As explained above, the anti-BoNT antibodies for this invention can beused for the in vivo or in vitro detection of BoNT toxin (e.g. BoNT/A1toxin) and thus, are useful in the diagnosis (e.g. confirmatorydiagnosis) of botulism. The detection and/or quantification of BoNT in abiological sample obtained from an organism is indicative of aClostridium botulinum infection of that organism.

The BoNT antigen can be quantified in a biological sample derived from apatient such as a cell, or a tissue sample derived from a patient. Asused herein, a biological sample is a sample of biological tissue orfluid that contains a BoNT concentration that may be correlated with andindicative of a Clostridium botulinum infection. Preferred biologicalsamples include blood, urine, saliva, and tissue biopsies.

Although the sample is typically taken from a human patient, the assayscan be used to detect BoNT antigen in cells from mammals in general,such as dogs, cats, sheep, cattle and pigs, and most particularlyprimates such as humans, chimpanzees, gorillas, macaques, and baboons,and rodents such as mice, rats, and guinea pigs.

Tissue or fluid samples are isolated from a patient according tostandard methods well known to those of skill in the art, most typicallyby biopsy or venipuncture. The sample is optionally pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, at physiological pH can be used.

A) Immunological Binding Assays

The BoNT polypeptide (e.g., BoNT/A1, BoNT/A2, etc.) can be detected inan immunoassay utilizing one or more of the anti-BoNA antibodies of thisinvention as a capture agent that specifically binds to the BoNTpolypeptide.

As used herein, an immunoassay is an assay that utilizes an antibody(e.g. a BoNT/A-neutralizing antibody) to specifically bind an analyte(e.g., BoNT/A). The immunoassay is characterized by the binding of oneor more anti-BoNT antibodies to a target (e.g. one or more BoNT/Asubtypes) as opposed to other physical or chemical properties toisolate, target, and quantify the BoNT analyte.

The BoNT marker can be detected and quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168, and the like) For areview of the general immunoassays, see also Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc.New York (1993); Basic and Clinical Immunology 7th Edition, Stites &Terr, eds. (1991)).

The immunoassays of the present invention can be performed in any of anumber of configurations (see, e.g., those reviewed in Maggio (ed.)(1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985)“Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers B.V.,Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: APractical Guide Academic Press, Orlando, Fla.; Price and Newman (eds.)(1991) Principles and Practice of Immunoassays Stockton Press, NY; andNgo (ed.) (1988) Non isotopic Immunoassays Plenum Press, NY).

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the capture agent and the analyte(e.g., a BoNT/A-neutralizing antibody/BoNT/A complex). The labelingagent can itself be one of the moieties comprising the antibody/analytecomplex. Thus, for example, the labeling agent can be a labeled BoNT/Apolypeptide or a labeled anti-BoNT/A antibody. Alternatively, thelabeling agent is optionally a third moiety, such as another antibody,that specifically binds to the BoNT antibody, the BoNT peptide(s), theantibody/polypeptide complex, or to a modified capture group (e.g.,biotin) which is covalently linked to BoNT polypepitde or to theanti-BoNT antibody.

In one embodiment, the labeling agent is an antibody that specificallybinds to the anti-BoNT antibody. Such agents are well known to those ofskill in the art, and most typically comprise labeled antibodies thatspecifically bind antibodies of the particular animal species from whichthe anti-BoNT antibody is derived (e.g., an anti-species antibody).Thus, for example, where the capture agent is a human derivedBoNT/A-neutralizing antibody, the label agent may be a mouse anti-humanIgG, i.e., an antibody specific to the constant region of the humanantibody.

Other proteins capable of specifically binding immunoglobulin constantregions, such as streptococcal protein A or protein G are also used asthe labeling agent. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see generally Kronval, et al., (1973) J. Immunol.,111:1401-1406, and Akerstrom, et al., (1985) J. Immunol., 135:2589-2542,and the like).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays are carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 5° C. to 45° C.

1) Non Competitive Assay Formats.

Immunoassays for detecting BoNT neurotoxins (e.g. BoNT serotypes and/orsubtypes) are, in certain embodiments, either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte (in this case, BoNT polypeptide) is directlymeasured. In one preferred “sandwich” assay, for example, the captureagent (e.g., an anti-BoNT antibody) is bound directly or indirectly to asolid substrate where it is immobilized. These immobilized anti-BoNTantibodies capture BoNT polypeptide(s) present in a test sample (e.g., ablood sample). The BoNT polypeptide(s) thus immobilized are then boundby a labeling agent, e.g., a BoNT/A-neutralizing antibody bearing alabel. Alternatively, the second antibody may lack a label, but it may,in turn, be bound by a labeled third antibody specific to antibodies ofthe species from which the second antibody is derived. Free labeledantibody is washed away and the remaining bound labeled antibody isdetected (e.g., using a gamma detector where the label is radioactive).

2) Competitive Assay Formats.

In competitive assays, the amount of analyte (e.g., BoNT/A) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte displaced (or competed away) from a capture agent(e.g., BoNT/A-neutralizing antibody) by the analyte present in thesample. For example, in one competitive assay, a known amount of BoNT/Ais added to a test sample with an unquantified amount of BoNT/A, and thesample is contacted with a capture agent, e.g., a BoNT/A-neutralizingantibody that specifically binds BoNT/A. The amount of added BoNT/A thatbinds to the BoNT/A-neutralizing antibody is inversely proportional tothe concentration of BoNT/A present in the test sample.

The BoNT/A-neutralizing antibody can be immobilized on a solidsubstrate. The amount of BoNT/A bound to the BoNT/A-neutralizingantibody is determined either by measuring the amount of BoNT/A presentin an BoNT/A-BoNT/A-neutralizing antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed BoNT/A.

B) Reduction of Non Specific Binding.

One of skill will appreciate that it is often desirable to reduce nonspecific binding in immunoassays and during analyte purification. Wherethe assay involves, for example BoNT/A polypeptide(s),BoNT/A-neutralizing antibody, or other capture agent(s) immobilized on asolid substrate, it is desirable to minimize the amount of non specificbinding to the substrate. Means of reducing such non specific bindingare well known to those of skill in the art. Typically, this involvescoating the substrate with a proteinaceous composition. In particular,protein compositions such as bovine serum albumin (BSA), nonfat powderedmilk, and gelatin are widely used.

C) Substrates.

As mentioned above, depending upon the assay, various components,including the BoNT polypeptide(s), anti-BoNT antibodies, etc., areoptionally bound to a solid surface. Many methods for immobilizingbiomolecules to a variety of solid surfaces are known in the art. Forinstance, the solid surface may be a membrane (e.g., nitrocellulose), amicrotiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube(glass or plastic), a dipstick (e.g., glass, PVC, polypropylene,polystyrene, latex, and the like), a microcentrifuge tube, or a glass,silica, plastic, metallic or polymer bead. The desired component may becovalently bound, or noncovalently attached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, e.g., as laminates, to obtain various properties. For example,protein coatings, such as gelatin can be used to avoid non specificbinding, simplify covalent conjugation, enhance signal detection or thelike.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas, (1970) J. Biol. Chem.245 3059.

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components. Alternatively, the surface is designed suchthat it nonspecifically binds one component but does not significantlybind another. For example, a surface bearing a lectin such asconcanavalin A will bind a carbohydrate containing compound but not alabeled protein that lacks glycosylation. Various solid surfaces for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082.

D) Other Assay Formats

BoNT polypeptides or anti-BoNT antibodies (e.g. BoNT/A neutralizingantibodies) can also be detected and quantified by any of a number ofother means well known to those of skill in the art. These includeanalytic biochemical methods such as spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.

Western blot analysis and related methods can also be used to detect andquantify the presence of BoNT polypeptides in a sample. The techniquegenerally comprises separating sample products by gel electrophoresis onthe basis of molecular weight, transferring the separated products to asuitable solid support, (such as a nitrocellulose filter, a nylonfilter, or derivatized nylon filter), and incubating the sample with theantibodies that specifically bind either the BoNT polypeptide. Theantibodies specifically bind to the biological agent of interest on thesolid support. These antibodies are directly labeled or alternativelyare subsequently detected using labeled antibodies (e.g., labeled sheepanti-human antibodies where the antibody to a marker gene is a humanantibody) which specifically bind to the antibody which binds the BoNTpolypeptide.

Other assay formats include liposome immunoassays (LIAs), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,(1986) Amer. Clin. Prod. Rev. 5:34-41).

E) Labeling of Anti-BoNT (e.g. BoNT/A-Neutralizing) Antibodies.

Anti-BoNT antibodies can be labeled by an of a number of methods knownto those of skill in the art. Thus, for example, the labeling agent canbe, e.g., a monoclonal antibody, a polyclonal antibody, a protein orcomplex such as those described herein, or a polymer such as an affinitymatrix, carbohydrate or lipid. Detection proceeds by any known method,including immunoblotting, western analysis, gel-mobility shift assays,tracking of radioactive or bioluminescent markers, nuclear magneticresonance, electron paramagnetic resonance, stopped-flow spectroscopy,column chromatography, capillary electrophoresis, or other methods whichtrack a molecule based upon an alteration in size and/or charge. Theparticular label or detectable group used in the assay is not a criticalaspect of the invention. The detectable group can be any material havinga detectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and, in general, anylabel useful in such methods can be applied to the present invention.Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., LacZ, CAT, horse radishperoxidase, alkaline phosphatase and others, commonly used as detectableenzymes, either as marker gene products or in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, ease of conjugation of thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detectedsimply by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence ofBoNT peptides. In this case, antigen-coated particles are agglutinatedby samples comprising the target antibodies. In this format, none of thecomponents need be labeled and the presence of the target antibody isdetected by simple visual inspection.

IX. Pharmaceutical Compositions.

The BoNT-neutralizing antibodies of this invention are useful inmitigating the progression of botulism produced, e.g., by endogenousdisease processes or by chemical/biological warfare agents. Typicallycompositions comprising one or preferably two or more differentantibodies are administered to a mammal (e.g., to a human) in needthereof.

We have discovered that particularly efficient neutralization of abotulism neurotoxin (BoNT) subtype is achieved by the use ofneutralizing antibodies that bind two or more subtypes of the particularBoNT serotype with high affinity. While this can be accomplished byusing two or more different antibodies directed against each of thesubtypes, this is less effective, inefficient and not practical. A BoNTtherapeutic is desirably highly potent, given the high toxicity of BoNT.Since it is generally necessary to use multiple antibodies to neutralizea given BoNT serotype with the required potency (see below and FIGS. 5,6, 16, and 17), the number of antibodies required would be prohibitivefrom a manufacturing standpoint if it were necessary to use differentantibodies for each subtype. Increasing the number of antibodies in themixture also reduces the potency, thus if in a mixture of fourantibodies, two neutralize A1 and two neutralize A2 toxin, then only 50%of the antibody will neutralize a given toxin. In contrast a mixture oftwo antibodies both of which neutralize A1 and A2 toxins will have 100%activity against either toxin and will be simpler to manufacture. Forexample for two BoNT/A subtypes (A1, A2) potent neutralization can beachieved with two to three antibodies. If different antibodies wererequired for BoNT/A1 and BoNT/A2 neutralization, then four to sixantibodies would be required. The complexity increases further foradditional subtypes. Thus in certain embodiments this invention providesfor compositions comprising neutralizing antibodies that bind two ormore BoNT subtypes (e.g., BoNT/A1, BoNT/A2, BoNT/A3, etc.) with highaffinity.

It was also a surprising discovery that when one starts combiningneutralizing antibodies that the potency of the antibody combinationincreases dramatically. This increase makes it possible to generate abotulinum antibody of the required potency for therapeutic use. It wasalso surprising that as one begins combining two and three monoclonalantibodies, the particular BoNT epitope that is recognized becomes lessimportant Thus for example, as indicated in Example 5, antibodies thatbind to the translocation domain and/or catalytic domains of BoNT hadneutralizing activity, either when combined with each other or whencombined with a mAb recognizing the BoNT receptor binding domain (HC)were effective in neutralizing BoNT activity. Thus, in certainembodiments, this invention contemplates compositions comprising atleast two, more preferably at least three high affinity antibodies thatbind non-overlapping epitopes on the BoNT.

In certain embodiments, this invention contemplates compositionscomprising two or more, preferably three or more different antibodiesselected from the group consisting of 3D12, RAZ1, CR1, ING1, ING2, an/orantibodies comprising one or more CDRs from these antibodies, and/or oneor more antibodies comprising mutants of these antibodies, such as the1D11, 2G11, and 5G4 mutants of ING1.

The BoNT-neutralizing antibodies of this invention are useful forparenteral, topical, oral, or local administration, such as by aerosolor transdermally, for prophylactic and/or therapeutic treatment. Thepharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. The antibodies comprising thepharmaceutical compositions of this invention, when administered orally,are preferably protected from digestion. This is typically accomplishedeither by complexing the antibodies with a composition to render themresistant to acidic and enzymatic hydrolysis or by packaging theantibodies in an appropriately resistant carrier such as a liposome.Means of protecting proteins from digestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of oneor more BoNT-neutralizing antibody dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of BoNT/A-neutralizing antibody in these formulations canvary widely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom about 1 mg up to about 200 mg per patient per day can be used.Methods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa. (1980).

The compositions containing the BoNT-neutralizing antibodies of thisinvention or a cocktail thereof are generally administered fortherapeutic treatments. Preferred pharmaceutical compositions areadministered in a dosage sufficient to neutralize (mitigate oreliminate) the BoNT toxin(s) (i.e., reduce or eliminate a symptom ofBoNT poisoning (botulism)). An amount adequate to accomplish this isdefined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the antibodies of this invention to effectivelytreat the patient.

X. Kits for Diagnosis or Treatment.

In another embodiment, this invention provides for kits for thetreatment of botulism or for the detection/confirmation of a Clostridiumbotulinum infection. Kits will typically comprise one or more anti-BoNTantibodies (e.g., BoNT-neutralizing antibodies for pharmaceutical use)of this invention. For diagnostic purposes, the antibody(s) canoptionally be labeled. In addition the kits will typically includeinstructional materials disclosing means of use BoNT-neutralizingantibodies in the treatment of symptoms of botulism. The kits may alsoinclude additional components to facilitate the particular applicationfor which the kit is designed. Thus, for example, where a kit containsone or more anti-BoNT antibodies for detection of diagnosis of BoNTsubtype, the antibody can be labeled, and the kit can additionallycontain means of detecting the label (e.g. enzyme substrates forenzymatic labels, filter sets to detect fluorescent labels, appropriatesecondary labels such as a sheep anti-human antibodies, or the like).The kits may additionally include buffers and other reagents routinelyused for the practice of a particular method. Such kits and appropriatecontents are well known to those of skill in the art.

In certain embodiments, kits provided for the treatment of botulismcomprise one or more BoNT neutralizing antibodies. The antibodies can beprovided separately or mixed together. Typically the antibodies will beprovided in a sterile pharmacologically acceptable excipient. In certainembodiments, the antibodies can be provided pre-loaded into a deliverydevice (e.g., a disposable syringe).

The kits can optionally include instructional materials teaching the useof the antibodies, recommended dosages, contraindications, and the like.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters that can be changed or modified to yieldessentially similar results.

Example 1 Preparation of Botulinum Neurotoxin Neutralizing Antibodies

Materials and Methods

A) Oligonucleotide Design.

Family-specific murine V_(H) and V_(K) primers were designed aspreviously described for human V-gene primers (Marks, et al. (1991) J.Mol. Biol. 222:581-597; Marks, et al., Eur. J. Immunol. 21:985-991) toamplify full-length rearranged V genes. Briefly, murine V_(H) and V_(K)DNA sequences were collected from the Kabat (Kabat, et al. (1991)Sequences of proteins of immunological interest, U.S. Department ofHealth and Human Services, U.S. Government Printing Office, Bethesda,Md.) and GenBank databases, aligned, and classified by family, andfamily-specific primers were designed to anneal to the first 23nucleotides comprising framework 1. Similarly, J_(H) and J_(K)gene-segment specific primers were designed to anneal to the final 24nucleotides comprising each of the 4 J_(H) and 5 J_(K) gene segments(Kabat, et al. supra.).

B) Vector Construction.

To construct the vector pSYN3, a 1.5 kb stuffer fragment was amplifiedfrom pCANTAB5E (Pharmacia Biotech, Milwaukee, Wis.) using PCR with theprimers LMB3 (Marks, et al. (1991) Eur. J. Immunol. 21:985-991) andE-tagback (5′-ACC ACC GAA TTC TTA TTA ATG GTG ATG ATG GTG GAT GAC CAGCCG GTT CCA GCG G-3′, (SEQ ID NO:137). The DNA fragment was digestedwith SfiI and Notf, gel purified, and ligated into pCANTAB5E digestedwith SfiI and NotI. Ligated DNA was used to transform Escherichia coliTGI (Gibson (1991) Studies on the Epstein-Barr virus genome. Universityof Cambridge, Cambridge, U.K.), and clones containing the correct insertwere identified by DNA sequencing. The resulting vector permitssubcloning of phage-displayed scFv as SfiI-NotI or Mcol-NotI fragmentsfor secretion into the periplasm of E. coli as native scFv with aC-terminal E epitope tag followed by a hexahistidine tag.

C) Immunizations.

For construction of library 1, BALB/c mice (16 to 22 g) were immunizedat 0, 2, and 4 weeks with pure BoNT/A H_(c) (Ophidian Pharmaceuticals,Madison, Wis.). Each animal was given subcutaneously 1 μg of materialadsorbed onto alum (Pierce Chemical Co., Rockford, Ill.) in a volume of0.5 ml. Mice were challenged 2 weeks after the second immunization with100,000 50% lethal doses of pure BoNT/A and were sacrificed 1 weeklater.

For construction of library 2, CD-1 mice (16 to 22 g) were immunized at0, 2, and 4 weeks with pure BoNT/A H_(c) and were sacrificed two weeksafter the third immunization. For both libraries, the spleens wereremoved immediately after sacrifice and total RNA was extracted by themethod of Cathala et al. (1993) DNA 2: 329.

D) Library Construction.

First-strand cDNA was synthesized from approximately 10 μg of total RNAas previously described in Marks, et al. (1991) J. Mol. Biol.222:581-597, except that immunoglobulin mRNA was specifically primedwith 10 pmol each of oligonucleotides MIgG1 For, MIgG3 For, and MC_(K)For (Table 3). For construction of library 1, rearranged V_(H), andV_(K) genes were amplified from first-strand cDNA by using commerciallyavailable V_(H) and V_(K) back primers and J_(H) and J_(K) forwardprimers (Recombinant Phage Antibody System; Pharmacia Biotech). Forlibrary 2, equimolar mixtures of family-specific V_(H) and V_(K) backprimers were used in conjunction with equimolar mixtures of J_(H) orJ_(K) gene-segment-specific forward primers in an attempt to increaselibrary diversity (see “Oligonucleotide design” above). Re-arrangedV_(H) and V_(K) genes were amplified separately in 50-μl reactionmixtures containing 5 μl of the first-strand CDNA reaction mixture, 20pmol of an equimolar mixture of the appropriate back primers, 20 pmol ofan equimolar mixture of the appropriate forward primers, 250 μm (each)deoxynucleoside triphosphate, 1.5 mm MgCI₂, 10 μg of bovine serumalbumin/ml, and 1 μl (5 U) of Thermus aquaticus (Taq) DNA polymerase(Promega) in the buffer supplied by the manufacturer. The reactionmixture was overlaid with paraffin oil (Sigma) and cycled 30 times (at95° C. for 1 min, 60° C. for 1 min, and 72° C. for 1 min). Reactionproducts were gel purified, isolated from the gel by using DEAEmembranes, eluted from the membranes with high-salt buffer, ethanolprecipitated, and resuspended in 20 μL of water (Sambrook, et al. (1989)Molecular cloning; a laboratory manual, 2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

TABLE 3 Oligonucleotide primers used for PCR of mouse immunoglobulingenes. Seq I.D. Primer ID Sequence No. A. 1st strand cDNA synthesisMouse heavy chain constant region primers MIgG1/2 For 5′ CTG GAC AGG GATCCA GAG TTC CA 3′ 138 MIgG3 For 5′ CTG GAC AGG GCT CCA TAG TTC CA 3′ 139Mouse _(κ )constant region primer MC_(κ)For 5′ CTC ATT CCT GTT GAA GCTCTT GAC 3′ 140 B. Primary PCR Mouse V_(H) back primers VH1 Back 5′ GAGGTG CAG CTT CAG GAG TCA GG 3′ 141 VH2 Back 5′ GAT GTG CAG CTT CAG GAGTCR GG 3′ 142 VH3 Back 5′CAG GTG CAG CTG AAG SAG TCA GG 3′ 143 VH4/6Back 5′ GAG GTY CAG CTG CAR CAR TCT GG 3′ 144 VH5/9 Back 5′ CAG GTY CARCTG CAG CAG YCT GG 3′ 145 VH7 Back 5′ GAR GTG AAG CTG GTG GAR TCT GG 3′146 VH8 Back 5′ GAG GTT CAG CTT CAG CAG TCT GG 3′ 147 VH10 Back 5′ GAAGTG CAG CTG KTG GAG WGT GG 3′ 148 VH11 Back 5′ CAG ATC CAG TTG CTG CAGTCT GG 3′ 149 Mouse V_(H) back primers VH1 Back 5′ GAC ATT GTG ATG WCACAG TCT CC 3′ 150 VH2 Back 5′ GAT GTT KTG ATG ACC CAA ACT CC 3′ 151 VH3Back 5′ GAT ATT GTG ATR ACB CAG GCW GC 3′ 152 VH4 Back 5′ GAC ATT GTGCTG ACM CAR TCT CC 3′ 153 VH5 Back 5′ SAA AWT GTK CTG ACC CAG TCT CC 3′154 VH6 Back 5′ GAY ATY VWG ATG ACM CAG WCT CC 3′ 155 VH7 Back 5′ CAAATT GTT CTC ACC CAG TCT CC 3′ 156 VH8 Back 5′ TCA TTA TTG CAG GTG CTTGTG GG 3′ 157 Mouse Jh forward primers JH1 For 5′ TGA GGA GAC GGT GACCGT GGT CCC 3′ 158 JH2 For 5′ TGA GGA GAC TGT GAG AGT GGT GCG 3′ 159 JH3For 5′ TGC AGA GAC AGT GAC CAG AGT CCC 3′ 160 JH4 For 5′ TGA GGA GAC GGTGAC TGA GGT TCC 3′ 161 Mouse Jκ forward primers: Jκ1 For 5′ TTT GAT TTCCAG CTT GGT GCC TCC 3′ 162 Jκ2 For 5′ TTT TAT TTC CAG CTT GGT CCC CCC 3′163 Jκ3 For 5′ TTT TAT TTC CAG TCT GGT CCC ATC 3′ 164 Jκ4 For 5′ TTT TATTTC CAA CTT TGT CCC CGA 3′ 165 Jκ5 For 5′ TTT CAG CTC CAG CTT GGT CCCAGC 3′ 166 C. Reamplification primers containing restriction sites MouseVH Sfi back primers VH1 Sfi 5′ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATGGCC GAG GTG 167 CAG CTT CAG GAG TCA GG 3′ VH2 Sfi 5′ GTC CTC GCA ACT GCGGCC CAG CCG GCC ATG GCC GAT GTG 168 CAG CTT CAG GAG TCR GG 3′ VH3 Sfi5′ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG 169 CAG CTG SAGSAG TCA GG 3′ VH4/6 Sfi 5′ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCCGAG GTY 170 CAG CTG CAR CAR TCT GG 3′ VH5/9 Sfi 5′ GTC CTG GCA ACT GCGGCC CAG CCG GCC ATG GCC CAG GTY 171 CAR CTG CAG CAG YCT GG 3′ VH7 Sfi5′ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC GAR GTG 172 AAG CTG GTGGAR TCT GG 3′ VH8 Sfi 5′ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC GAGGTT 173 CAG CTT CAG CAG TCT GG 3′ VH10 Sfi 5′ GTC CTC GCA ACT GCG GCCCAG CCG GCC ATG GCC GAA GTG 174 CAG CTG KTG GAG WCT GG 3′ VH11 Sfi5′ GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG ATC 175 CAG TTG CTGCAG TCT GG 3′ D Mouse Jκ Not forward primers Jκ1 Not 5′ GAG TCA TTC TCGACT TGC GGC CGC TTT GAT TTC CAG CTT 176 GGT GCC TCC 3′ Jκ2 Not 5′ GAGTCA TTC TCG ACT TGC GGC CGC TTT TAT TTC CAG CTT 177 GGT CCC CCC 3′ Jκ3Not 5′ GAG TCA TTC TCG ACT TGC GGC CGC TTT TAT TTC CAG TCT 178 GGT CCCATC 3′ Jκ4 Not 5′ GAG TCA TTC TCG ACT TGC GGC CGC TTT TAT TTC CAA CTT179 TGT CCC CGA 3′ Jκ5 Not 5′ GAG TCA TTC TCG ACT TGC GGC CGC TTT CAGCTC CAG CTT 180 GGT CCC AGC 3′ R = A/G, Y = G/T, S = G/C, K = G/T, W= A/T, M = A/C, V = C/G/A, B = G/C/T, and H = C/A/T.

ScFv gene repertoires were assembled from purified V_(H) and V_(K) generepertoires and linker DNA by using splicing by overlap extension.Linker DNA encoded the peptide sequence (Gly₄Ser₃, (SEQ ID NO:181)Huston, et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) and wascomplementary to the 3′ ends of the rearranged V_(H) genes and the 5′ends of the rearranged V. genes. The V_(H) and V_(K) DNAs (1.5 μg ofeach) were combined with 500 ng of linker DNA (Recombinant PhageAntibody System; Pharmacia Biotech) in a 25 μl PCR mixture containing250 μm (each) deoxynucteoside triphosphate, 1.5 mM MgCl, 10 μg of bovineserum albumin/ml, and 1 μl (5 U) of Taq DNA polymerase (Promega) in thebuffer supplied by the manufacturer, and the mixture was cycled 10 times(at 94° C. for 1 min, 62° C. for 1 min, and 72° C. for 1 min) to jointhe fragments. Flanking oligonucleotide primers (RS, provided in theRecombinant Phage Antibody System kit, for library I and an equimolarmixture of V_(H) Sfi and JKNot primers [Table 3] for library 2) wereadded, and the reaction mixture was cycled for 33 cycles (at 94° C. for1 min, 55° C. for 1 min, and 72° C. for 1 min) to append restrictionsites.

ScFv gene repertoires were gel purified as described above, digestedwith Sfil and Notl, and purified by electroelution, and 1 μg of eachrepertoire was ligated into either 1 μg of pCANTAB5E vector (PharmaciaBiotech) (library 1) or 1 μg of pHEN-1 (Hoogenboom, et al. (1991)Nucleic Acids Res. 19: 4133-4137) (library 2) digested with Sfil andNotl. The ligation mix was purified by extraction withphenol-chloroform, ethanol precipitated, resuspended in 20 μl of water,and 2.5 μl samples were electroporated (Dower, et al. (1988) NucleicAcids Res. 16:6127-6145) into 50 μl of E. coli TGI (Gibson (1984),Studies on the Epstein-Barr virus genome. University of Cambridge,Cambridge, U.K.). Cells were grown in 1 ml of SOC (Sambrook, et al.supra.) for 30 min and then plated on TYE (Miller (1972) Experiments inmolecular genetics., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) medium containing 100 μg of AMP/ml and 1% (wt/vol)GLU(TYE-AMP-GLU). Colonies were scraped off the plates into 5 ml of 2×TY broth (Miller (1972) supra.) containing 100 μg of AMP/ml, 1% GLU (2×TY-AMP-GLU), and 15% (vol/vol) glycerol for storage at −70° C. Thecloning efficiency and diversity of the libraries were determined by PCRscreening (Gussow, et al. (1989), Nucleic Acids Res. 17: 4000) asdescribed by Marks et al. (1991) Eur. J. Immunol., 21: 985-991.

E) Preparation of Phage.

To rescue phagemid particles from the libraries, 10 ml of 2×. TY-AMP-GLUwas inoculated with an appropriate volume of bacteria (approximately 50to 100 μl) from the library stocks to give an A₆₀₀ of 0.3 to 0.5 andbacteria were grown for 30 min with shaking at 37° C. About 10¹² PFU ofVCS-M13 (Stratagene) particles were added, and the mixture was incubatedat overnight at 4° C. Tubes were blocked for 1 h at 37° C. with 2% MPBS,and selection, washing, and elution were performed exactly as describedin reference 35 by using phage at a concentration of 5.0×10¹² TU/ml.One-third of the eluted phage was used to infect 10 ml of log-phase E.coli TGI, which was plated on TYE-AMP-GLU plates as described above.

The rescue-selection-plating cycle was repeated three times, after whichclones were analyzed for binding by ELISA. Libraries were also selectedon soluble BoNT/A H_(c). For library 1, 1.0 mg of BoNT/A H_(c) (700μg/ml) was biotinylated (Recombinant Phage Selection Module; Pharmacia)and purified as recommended by the manufacturer. For each round ofselection, 1 ml of phage (approximately 10¹³ TU) were mixed with 1 ml ofPBS containing 4% skim milk powder, 0.05% Tween 20, and 10 μg ofbiotinylated BoNT/A H_(c)/ml. After 1 h at room temperature,antigen-bound phage were captured on blocked streptavidin-coated M280magnetic beads (Dynabeads; Dynal) as described by Schier et al. (1996)J. Mol. Biol., 255: 28-43. Dynabeads were washed a total of 10 times(three times in TPBS, twice in TMPBS, twice in PBS, once in MPBS, andtwo more times in PBS). Bound phage were eluted from the Dynabeads byincubation with 100 μl of 100 mM triethylamine for 5 min and wereneutralized with 1 M Tris-HCl, pH 7.5, and one-third of the eluate wasused to infect log-phase E. coli TG1.

For library 2, affinity-driven selections (Hawkins, et al. (1992) J.Mol. Biol. 226: 889-896; Schier, et al. (1996) supra.)) were performedby decreasing the concentration of soluble BoNT/A H_(c) used forselection (10 μg/ml for round 1, 1 μg/ml for round 2, and 10 ng/ml forround 3). Soluble BoNT/A H_(c) was captured on 200 μl of Ni²⁺-NTA(Qiagen) via a C-terminal hexahistidine tag. After capture, the Ni²⁺-NTAresin was washed a total of 10 times (5 times in TPBS and 5 times inPBS), bound phage were eluted as described above, and the eluate wasused to infect log-phase E. coli TGI.

F) Initial Characterization of Binders.

Initial analysis for binding to BoNT/A, BoNT/A H_(c), and BoNT/A H_(N)(Chen, et al. (1997) Infect. Immun. 65: 1626-1630) was performed byELISA using bacterial supernatant containing expressed scFv. Expressionof scFv (De Bellis, et al., (1990) Nucleic Acids Res. 18: 1311) wasperformed in 96-well microtiter plates as described by marks et al.(1991) J. Mol. Biol., 222: 581-597. For ELISA, microtiter plates (Falcon3912) were coated overnight at 4° C. with either BoNT/A, BoNT/A H_(c),or BoNT/A H_(N) (10 μg/ml) in PBS and then were blocked with 2% MPBS for1 h at room temperature. Bacterial supernatants containing expressedscFv were added to wells and incubated at room temperature for 1.5 h.Plates were washed six times (3 times with TPBS and 3 times with PBS),and binding of scFv was detected via their C-terminal peptide tags (Eepitope tag for library 1 in pCANTAB5E and myc epitope tag [Munro, etal. (1986) Cell 46: 291-300] for library 2 in pHEN-1) by using eitheranti-myc tag antibody (9E10; Santa Cruz Biotechnology) or anti-Eantibody (Pharmacia Biotech) and peroxidase-conjugated anti-mouse Fcantibody (Sigma), as described by Marks et al. (1991) J. Mol. Biol.,222: 581-597 and Schier et al. (1996) Gene 169: 147-155. The number ofunique binding scFv was determined by BstN1 fingerprinting and DNAsequencing.

G) Subcloning, Expression, and Purification of scFv.

To facilitate, purification, scFv genes were subcloned into theexpression vector pUC119mycHis (Schier et al. (1995) J. Mol. Biol., 263:551-567) or pSYN3, resulting in the addition of a hexahistidine tag atthe C-terminal end of the scFv. Two hundred-milliliter cultures of E.coli TG1 harboring one of the appropriate phagemids were grown,expression of scFv was induced with IPTG (De Bellis, et al. (1990),Nucleic Acids Res. 18:1311), and the cultures were grown at 25° C.overnight. scFv was harvested from the periplasm (Breitling, et al.(1991) Gene 104:147-153), dialyzed overnight at 4° C. against IMACloading buffer (50 mM sodium phosphate [pH 7.5], 500 mM NaCl, 20 mMimidazole), and then filtered through a 0.2-μm-pore-size filter. scFvwas purified by IMAC (Hochuli, et al. (1988) Bio/Technology 6:1321-1325) as described by Schier et al. (1995) supra.

To separate monomeric scFv from dimeric and aggregated scFv, sampleswere concentrated to a volume of <1 ml in a centrifugal concentrator(Centricon 10; Amicon) and fractionated on a Superdex 75 column(Pharmacia) by using HBS. The purity of the final preparation wasevaluated by assaying an aliquot by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Protein bands were detectedby Coomassie blue staining. The concentration was determinedspectrophotometrically, on the assumption that an A₂₈₀ of 1.0corresponds to an scFv concentration of 0.7 mg/ml.

H) Measurement of Affinity and Binding Kinetics.

The K_(d)s of purified scFv were determined by using surface plasmonresonance in a BIAcore (Pharmacia Biosensor AB). In a BIAcore flow cell,approximately 600 RU of BoNT/A H_(c) (15 μg/ml in 10 mM sodium acetate[pH 4.5]) was coupled to a CM5 sensor chip by usingN-hydroxysuccinimide-N-ethyl-N′-(dimethylaminopropyl) carbodimidechemistry (Johnson, et al. (1991) Anal. Biochem. 198: 268-277). Thisamount of coupled BoNT/A H_(c) resulted in a maximum RU of 100 to 175 ofscFv bound. For regeneration of the surface after binding of scFv, 5 μlof 4 M MgCl₂ was injected, resulting in a return to baseline. Thesurface was reused 20 to 30 times under these regeneration conditions.Association was measured under a continuous flow of 5 μl/min with aconcentration range from 50 to 1,000 nM. k_(on) was determined from aplot of In (dR/dt)/t versus concentration, where R is response and t istime (Karlsson, et al. (1991) J. Immunol. Methods 145: 229-240). k_(off)was determined from the dissociation part of the sensorgram at thehighest concentration of scFv analyzed (Karlsson, et al. (1991) J.Immunol. Methods 145: 229-240) by using a flow rate of 30 μl/min. K_(d)was calculated as k_(off)/k_(on).

I) Epitope Mapping.

Epitope mapping was performed by using surface plasmon resonance in aBIAcore. In a BIAcore flow cell, approximately 1,200 RU of BoNT/A H_(c)was coupled to a CM5 sensor chip as described above. With a flow rate of5 μl/min, a titration of 100 nM to 1 μM scFv was injected over the flowcell surface for 5 min to determine an scFv concentration which resultedin near saturation of the surface. Epitope mapping was performed withpairs of scFv at concentrations resulting in near saturation and atleast 100 RU of scFv bound. The amount of scFv bound was determined foreach member of a pair, and then the two scFv were mixed together to givea final concentration equal to the concentration used for measurementsof the individual scFv. scFv recognizing different epitopes showed anadditive increase in the RU bound when injected together (FIG. 2 panelA), while scFv recognizing identical epitopes showed only a minimalincrease in RU (FIG. 2 panel B).

J) In Vitro Neutralization Studies.

In vitro neutralization studies were performed by using a mousehemidiaphragm preparation, as described by Deshpande et al. (1995)Toxicon 33: 551-557. Briefly, left and right phrenic nerve hemidiaphragmpreparations were excised from male CD/I mice (25 to 33 g) and suspendedin physiological solution (135 mM NaCl, 5 mM KCl, 15 mM NaHCO₃, 1 mMNa2HPO₄, 1 mM MgCl₂, 2 mM CaCl₂, and 11 mM GLU). The incubation bath wasbubbled with 95% O₂-5% CO₂ and maintained at a constant temperature of36° C. Phrenic nerves were stimulated supramaximally at 0.05 Hz withsquare waves of 0.2 ms duration. Isometric twitch tension was measuredwith a force displacement transducer (Model FT03; Grass) connected to achart recorder. Purified scFv were incubated with purified BoNT/A for 30min at room temperature and then added to the tissue bath, resulting ina final scFv concentration of 2.0×10⁻⁸ M and a final BoNT/Aconcentration of 2.0×10⁻¹¹ M. For each scFv studied, time to 50% twitchtension reduction was determined three times for BoNT/A alone and threetimes for scFv plus BoNT/A. The combination of S25 and C25 was studiedat a final concentration of 2.0×10⁻⁸ M each. Differences between timesto 50% twitch reduction were determined by a two-tailed t test, with a Pvalue of <0.05 considered significant.

TABLE 4 Frequency of binding of clones from phage antibody librariesFrequency of ELISA-positive clones^(a) in selection round: Antigen usedfor selection 1 2 3 Library 1^(b) BoNT/A: immunotube^(c) 20/184 124/184ND BoNT/A H_(c): immunotube 7/92 86/92 88/92 BoNT/A H_(c):biotinylated^(d) 7/90 90/90 90/90 14/48  48/48 ND Library 2^(e) BoNT/A:immunotube ND 81/92 ND BoNT/A H_(c): immunotube ND ND 76/92 BoNT/A H,:Ni²⁺-NTA^(f) ND ND 67/92 ^(a)Expressed as number of positiveclones/total number of clones. For selections on BoNT/A and BoNT/AH_(c), ELISA was done on immobilized BoNT/A and BoNT/A H_(c),respectively. ND, data not determined from selection performed.^(b)Derived from a mouse immunized twice with BoNT/A H_(c) and once withBoNT/A. ^(c)Immunotube selections were performed with the antigenabsorbed onto immunotubes. ^(d)Biotinylated selections were performed insolution with capture on streptavidin magnetic beads. ^(e)Derived from amouse immunized three times with BoNT/A H_(c). ^(f)Ni²⁺-NTA selectionswere performed in solution with capture on Ni²⁺-NTA agarose.Results.

A) Phase Antibody Library Construction and Characterization.

Two phage antibody libraries were constructed from the V_(H) and V_(K)genes of immunized mice (FIG. 1). For library 1, a mouse was immunizedtwice with BoNT/A H_(C) and challenged 2 weeks after the secondimmunization with 100,000 50% lethal doses of BoNT/A. The mouse survivedthe BoNT/A challenge and was sacrificed 1 week later. The spleen wasremoved immediately after sacrifice, and total RNA was prepared. Forlibrary construction, IgG heavy-chain and kappa light-chain mRNA werespecifically primed and first-strand cDNA was synthesized. V_(H) andV_(K) gene repertoires were amplified by PCR, and V_(H), J_(H) V_(K),and J_(K) primers were provided in the recombinant phage antibodysystem.

The V_(H) and V_(K) gene repertoires were randomly spliced together tocreate an scFv gene repertoire by using synthetic DNA encoding the15-amino-acid peptide linker (G₄S)₃ (SEQ ID NO:182). Each scFv generepertoire was separately cloned into the phage display vector pCANTAB5E(Pharmacia). After transformation, a library of 2.1×10⁶ members wasobtained. Ninety percent of the clones had an insert of the appropriatesize for an scFv gene, as determined by PCR screening, and the clonedscFv genes were diverse, as determined by PCR fingerprinting. DNAsequencing of 10 unselected clones from library 1 revealed that allV_(H) genes were derived from the murine V_(H)2 family and all V_(K)genes were derived from the murine V_(K)4 and V_(K)6 families (Kabat, etal. (1991) supra.). Based on this observed V-gene bias, family-specificV_(H) and V_(K) primers were designed along with J_(H) and J_(K)gene-segment-specific primers (Table 3). These primers were then used toconstruct a second phage antibody library.

For library 2, a mouse was immunized three times with BoNT/A H_(c) andsacrificed 2 weeks after the third immunization. The mouse was notchallenged with BoNT/A prior to spleen harvest, as this led to theproduction of non-H_(c)-binding antibodies (see “Selection and initialcharacterization of phage antibodies” below). The spleen was harvested,and a phage antibody library was constructed as described above, exceptthat V_(H)-, J_(H)-, V_(K)-, and J_(K)-specific primers were used. Aftertransformation, a library of 1.0×10⁶ members was obtained. Ninety-fivepercent of the clones had an insert of the appropriate size for an scFvgene, as determined by PCR screening, and the cloned scFv genes werediverse, as determined by PCR fingerprinting (data not shown). DNAsequencing of 10 unselected clones from library 2 revealed greaterdiversity than was observed in library 1; V_(H) genes were derived fromthe V_(H1), V_(K)2, and V_(K)3 families, and V_(K) genes were derivedfrom the V_(K)2, V_(K)3, V_(K)4, and V_(K)6 families (Kabat, et al.(1991) supra.).

B) Selection and Initial Characterization of Phage Antibodies.

To isolate BoNT/A binding phage antibodies, phage were rescued from thelibrary and selected on either purified BoNT/A or BoNT/A H_(c).Selections were performed on the holotoxin in addition to H_(c), sinceit was unclear to what extent the recombinant toxin H_(c) would mimicthe conformation of the H_(c) in the holotoxin. Selection for BoNT/A andBoNT/A H_(c) binders was performed on antigen adsorbed to polystyrene.In addition, H_(c) binding phage were selected in solution onbiotinylated H_(c), with capture on streptavidin magnetic beads (forlibrary 1) or on hexahistidine tagged H_(c), with capture on Ni²⁺-NTAagarose (for library 2). Selections in solution were utilized based onour previous observation that selection on protein adsorbed topolystyrene could yield phage antibodies that did not recognize nativeprotein (Schier et al. (1995) Immunotechnology, 1: 73-81). Selection insolution was not performed on the holotoxin due to our inability tosuccessfully biotinylate the toxin without destroying immunoreactivity.

After two to three rounds of selection, at least 67% of scFv analyzedbound the antigen used for selection (Table 2). The number of uniquescFv was determined by DNA fingerprinting followed by DNA sequencing,and the specificity of each scFv was determined by ELISA on pure BoNT/Aand recombinant BoNT/A H_(c) and HN scFv binding BoNT/A but not binding,H_(c) or HN were presumed to bind the light chain (catalytic domain). Atotal of 33 unique scFv were isolated from mice immunized with H_(c) andchallenged with BoNT/A (Table 5, library 1). When library 1 was selectedon holotoxin, 25 unique scFv were identified. Only 2 of these scFv,however, bound H_(c), with the majority (Hathaway, et al. (1984) J.Infect. Dis. 150:407-412) binding the light chain and 2 binding H_(N).The two H_(c) binding scFv did not express as well as other scFvrecognizing similar epitopes, and they were therefore not characterizedwith respect to affinity or neutralization capacity (see below).

Selection of library 1 on H_(c) yielded an additional eight unique scFv(Tables 3 and 4). Overall, however, only 50% of scFv selected on H_(c)also bound holotoxin. This result suggests that a significant portion ofthe H_(c) surface may be inaccessible in the holotoxin. Alternatively,scFv could be binding, H_(c) conformations that do not exist in theholotoxin. From mice immunized with H_(c) only (library 2), all scFvselected on holotoxin also bound H_(c). As with library 1, however, only50% of scFv selected on H_(c) bound holotoxin. In all, 18 unique H_(c)binding scFv were isolated from library 2, resulting in a total of 28unique H_(c) binding scFv (Tables 5 and 6). scFv of identical or relatedsequences were isolated on both H_(c) immobilized on polystyrene andH_(c) in solution. Thus, in the case of H_(c), the method of selectionwas not important.

TABLE 5 Specificity of BoNT binding scFv selected from phage antibodylibraries. Number of unique scFv scFv Specificity library 1 library 2BoNT/A H_(c) 10 18 BoNT/A H_(N) 2 0 BoNT/A light chain 21 0 Total 33 18

C) Epitope Mapping.

All 28 unique H_(c) binding scFv were epitope mapped using surfaceplasmon resonance in a BIAcore. Epitope mapping was performed with pairsof scFv at concentrations resulting in near saturation of the chipsurface and at least 100 RU of scFv bound. The amount of scFv bound wasdetermined for each member of a pair, and then the two scFv were mixedtogether to give a final concentration equal to the concentration usedfor measurements of the individual scFv. Those scFv recognizingdifferent epitopes showed an additive increase in the RU bound wheninjected together (FIG. 2, panel A), while scFv recognizing identicalepitopes showed only a minimal increase in RU (FIG. 2, panel B). By thistechnique, mapping of the 28 scFv yielded 4 nonoverlapping epitopesrecognized on H_(c) (Table 6). scFv recognizing only epitopes 1 and 2were obtained from library 1, whereas scFv recognizing all 4 epitopeswere obtained from library 2.

Many of the scFv recognizing the same epitope (C1 and S25; C9 and C15;1E8 and 1G7; 1B6 and 1C9; C25 and C39; 2G5, 3C3, 3F4, and 3H4; 1A1 and1F1; 1B3 and 1C6; 1G5 and 1H6; 1F3 and 2E8) had V_(H) domains derivedfrom the same V-D-J rearrangement, as evidenced by the high level ofhomology of the V_(H)CDR3 and V_(H)-gene segment (Table 6). These scFvdiffer only by substitutions introduced by somatic hypermutation or PCRerror. For epitopes 1 and 2, most or all of the scFv recognizing thesame epitope are derived from the same or very similar V_(H)-genesegments but differ significantly with respect to V_(H)CDR3 length andsequence (5 of 9 scFv for epitope 1; 8 of 8 scFv for epitope 2) (Table6). These include scFv derived from different mice. Given the greatdegree of diversity in V_(H)CDR2 sequences in the primary repertoire(Tomlinson et al. (1996) J. Mol. biol., 256: 813-817), specificV_(H)-gene segments may have evolved for their ability to form bindingsites capable of recognizing specific pathogenic antigenic shapes. Incontrast, greater structural variation appears to occur in therearranged Y_(K) genes. For example, three different germ line genes andCDR1 main-chain conformations (Chothia, et al. (1987) J. Mol. Biol. 196:901-917) are observed for epitope 21 where all the V_(H) (genes arederived from the same germ line gene. Such “promiscuity” in chainpairings has been reported previously (Clackson, et al. (1991) Nature352: 624-628).

D) Affinity, Binding Kinetics, and In Vitro Toxin Neutralization.

Affinity, binding kinetics, and in vitro toxin neutralization weredetermined for one representative scFv binding to each epitope. For eachepitope, the scFv chosen for further study had the best combination ofhigh expression level and slow k_(off), as determined during epitopemapping studies. K_(d) for the four scFv studied ranged between 7.3×10⁻⁸and 1.1×10⁻⁹ M (Table 7), values comparable to those reported formonoclonal IgG produced from hybridomas (Foote, et al. (1991) Nature352: 530-532). C25 has the highest affinity (K_(d)=1.1×10⁻⁹ M) reportedfor an anti-botulinum toxin antibody. k_(on) differed over 84-fold, andk^(off) differed over 33-fold, between scFv (Table 7). In vitro toxicneutralization was determined by using a mouse hemidiaphragm preparationand measuring the time to 50% twitch tension reduction for BoNT/A aloneand in the presence of 2.0×10⁻⁸ M scFv. Values are reported in time to50% twitch reduction. scFv binding to epitope 1 (S25) and epitope 2(C25) significantly prolonged the time to neuroparalysis: 1.5-fold(152%) and 2.7-fold (270%), respectively (Table 7 and FIG. 3). Incontrast, scFv binding to epitopes 3 and 4 had no significant effect onthe time to neuroparalysis. A mixture of S25 and C25 had a significantadditive effect on the time to neuroparalysis, with the time to 50%twitch reduction increasing 3.9-fold (390%).

TABLE 6 Deduced protein sequences of V_(H) and V_(L) of BoNT/A H_(C)binding scFv, classified by epitope recognized. TABLE. Deduced proteinsequences of V_(H) and V_(L) of BoNT/A H_(C) binding scFv, classified byepitope recognized Re- Epi- Sequence^(b) gion tope Clone Lib^(a)Framework 1 CDR 1 Framework 2 CDR 2 V_(H) 1 C15 1QVELQQSGAELVP.PGASVKLSCKTSGYSFT SYWMN WVKQGPGQGLEWIG MIHPSNSEIRFNQEFEDC9 1 ------------------------------- ----- ------------------------------H ID5 2 E---VE-------------N----A------ ---------R--------- --------T-L----K- C1 1 -----------------------A----------- ----R--------- -------DT-------- S25 1-----------------------A----L- ----- ----R--------- -----D-DT--------1B6 2 --Q------------V---I---A---T-I D-A-H ----S-AKS-----V-SSYYGDTDY--I-NG 1C9 2 --Q-K----------V---I---G---T-I D-AVH----SHAKS----- V-STYYGDADY-PK-KG 1E8 2 E-Q--E--PG--X-SQ-LS-T-TVT---I-D-AW- -IR-F--KK---M- Y-S YSGSTGYNPSLKS 1G7 2E-Q--E--PG -K-SQ-LS-T-TVT---I- D-AWY -IR-F--KK---M- Y-S YSGSTGYNPSLKS 21A1 2 EVKLVESGGGLVQPGGSRKLSCATSGFTFS DYYHS WIRQSPDKRLEWVATISDGGTYTYYPDSVKG 1F1 2 -----------------L-----A------ N-G---V--T--------- H--S--S-N--S----- C39 1 Q-Q-Q-----S-K----L-----A----------- -V--T-E------- ------S---------- C25 1Q-Q-Q-------K----L-----A------ ---Y -V--T-E------- ------S---------- 2G52 ------------K----L-----A------ S-A-- -V--T-E------- -----------T-N---3C3 2 ----K-------K----L-----A------ S-A-- -V--T-E------------------T-N--- 3F4 2 EG----------K----L-----A------ S-A---V--T-BH------ -------P---T-N--- 3H4 2 ------------K---PL-----A------S-A-- -V--T-Eh------ -------F---T-N--- 3 1B3 2EVQLQESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG1C6 2 QI--LQ------------------------ ----- ------------------------------- 2B6 2 VKLVESGP-L-EPSQSLSLTCTVTGYSIT- D-AWN-I--F--NK---MG Y-N-----N-NP -L-N 1G5 2 Q----Q--AHL----A-VKM--K---Y--T--WT --K-R--Q----IG D-YP-GSGSTNYNKF-S 1H6 2-----Q--AEL-K--A-VKM--K---Y--T --WT --K-R--Q----IG D-YP-SGSTNTNEKF-S 41F3 2 EVQLQQSGAELVKPGASVKLSCKASGYTPT SFWMH WVKGRPGRGLEWIGRLDPNSGBTKYNEKPKS 2E8 2 ------------------------------ ------------------- ------------K---- V_(L) 1 C15 1 DIELTQSPAIMSASFGEKVIMTCSASS     SVSHMY WYQQKPGSSPRLLIY DTSNLAS C9 1 --D---------S-------I------     ---Y-H -F-----T--KPW-- S------ 1D5 2 -----------A--------I------S   I-S-NLH -----SETSPKPW-- G------ C1 1 ---------------------------     ---Y-- --------------- ------- S25 1 ---------L-A--------I---V--S   I-S-NLH -----S-T--KPW-- G------ 1B6 2 ---------SLAV-1.-QRA-IS-RA-ESVDSYGN-F-H -------QP-K---- RA---E- 1C9 2 ---------SLAV-L-QRA-IS-RA-ESVDSYGN-P-H -------QP-K---- RA---E- 1E8 2 ---------------------------     ---Y-H -----S-T--KRW-- ---K--- 1G7 2 ---------------------------     ---Y-H -----S-T--KRW-- ---K--- 2 1A1 2 DIELTQSPASLAVSLGQRATISCEASESVDSYGNSFMH WYQQKPGQPPKLLIY LASNLRS 1F1 2 --------T----------------------------- --------------- ------- C39 1 ----------------R----------------H---- --------------- ------- C25 1 ---------------------------------H---Q --------------- R-----P 2G5 2 ---------IMSA-P-EKVTTT-S--S     SSV-Y-- -P-----TS-K-W-- ST---A- 3C3 2 ---------IMSA-P-EKVTTT-----------H---Q -F-----TS-K-W-- ST---A- 3F4 2 -T-------IMSA-P-EKVTMT-S--S     SV-Y-Y -------SS-R---- DT---A- 3H4 2 ---------IMSA-P-EKVTMT----S-   VSS-YL- -------SS-R---- DT---A- 3 1B3 2 DSELTQSPTTMAASPGEKITTTCSASSS   ISSSNYLH WYQQRPGFSPKLLIY RTSNLAS 1C6 2 -I------ASL-V-L-RRA--S-R--E-VEYYGTSLMQ ----K--QP------ AA--VE- 2B6 2 YI------ASL-V-L-QRA--S-R--E-VDSYGNSFM- ----K--QP------ LA---E- 1G5 2 -I------ASL-V-L-QRA--S-R--E-VEYYGTSLMQ ----K--QP------ AA--VE- 1H6 2 -I------AI-S------V-----V---   ---SN-- ----KS-T----W-- G------ 4 1F3 2 DIELTQSPASMSASPGEKVTMTCRATSS   VSSSYLH WYQQKSGASPKLWIY SASNLAS 2ES 2 --------TT-A------I-I--S-S--   IG-N--- -----P-F----L-- RT----- Re- Epi- Sequence^(b) gion topeClone Lib^(a) Framework 3 CDR 3 Framework 4 SEQ ID NO: V_(H) 1 C15 1MATLTVDKSSSTAYMQLSSPTSEDSAVYYCAR GIYYDYDGGNYYAMDY WGQGTTVTASS 373 C9 1-------------------------------- ----V----------- --------V-- 374 ID5 2K------------------------------- -------E-Y--TL-- ------L-V-- 375 C1 1K------R-----IH----------------- -L-GYGF    WYFDV --------V-- 376 S25 1K------T------------------------ -L-NGF     WYF-V --------V-- 377 1B6 2K---------N----E-APL--D---I----- RGKG        ---- --------V-- 378 1C9 2K-----N---N----B-PRL------I----- RGKG        ---- -----S--V-- 379 1E8 2RISI-R-T-KNQFFL--N-V-T--TGT----- -YD         ---- -----S--V-- 380 1G7 2RISI-R-T-KNQFFL--N-V-T--TGT----- -YD         ---- -----S--V-- 381 2 1A12 RFTISRDHNAKNTLYLQMSSLKSEDTAHYYCVR HGYGNYPSH  WYFDW WGAGTTVTVSS 382 1F12 -V--------S---------Q-------L-T- --------Y ----- 383 C39 1-----------N--------------I----- YR-DEGL       -Y ----------- 384 C25 1-----------N------------------S- YR-DDAM       -Y --Q-------- 385 2G5 2----------HN------H-----------A- NLPYDHV       -Y --Q--S----- 386 3C3 2----------HN------H-----------A- NLPYDHV       -Y --Q--S----- 387 3F4 2----------HN------H-----------A- NLPYDHV       -Y --Q--S----- 388 3H4 2----------HN------H-----------A- NLPYDHV       -Y --Q--S----- 389 3 1B32 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DWSEGYYYYG   MDV WGQGTTVIVSS 390 1C62 -------------------------------- ----------   --- ----------- 391 2B62 -ISIT--T---QFF-KL--VTS----T----- AGDGY-VD   WYFDV --T-------- 392 1G52 KA-LTV-T-SS-A-N-LS--TS--S------- ELGD        A--Y -----S----- 393 1H62 KA-LTV-T-SS-A-M-LS--TS--S------- ELGD        A--Y -----S----- 394 41F3 2 KATLTVDKPSSTAYMELSSLTSEDSAVYYCAR EAYGYWN      PDV WGTGTTVTVSS 3952E8 2 -------------------------------- -------      --- ----------- 396V_(L) 1 C15 1 GVPIRFSGSGSGTSYSLTISRHEAEDSATYYC QQWSSYPFT FGSGTKLELKR 397C9 1 ---A----------------SV----A----- --Y-G--L- --A-----I-- 398 1D5 2---V----------------S-----A----- ---G---L- --G-----I-- 399 C1 1---V----------------------A----- -------L- --A-------- 400 S25 1---V----------------S-----A----- -------L- --A-----I-- 401 1B6 2-I-A-------R-DFT--INPV--D-V----- --SNED-P- --A-------- 402 1C9 2-I-A-------R-DFT---NPV--D-V----- --SNED-Y- --G-----I-- 403 1E8 2---A----------------S-----A----- -----N-L- --A-------- 404 1G7 2---A----------------S-----A----- -----N-L- --A-------- 405 2 1A1 2GVPARFSGSGSRTDFTLTIDPVEADDAATYYC QQNNEDPYT FGGGTKLSIKR 406 1F1 2-------------------------------- --------- ----------- 407 C39 1-------------------------------- --------- ----------- 408 C25 1-I---------G-------N------V----- --S----F- --S-------- 409 2G5 2-----------G-SYS---SRM--E------- --RSSY--- ----DQAGN-S 410 3C3 2-----------G-SYS---SRM--E------- --RSSY--- ----DQAGN-- 411 3F4 2---V-------G-SYS---SRM--E------- --WSSY-P- ----------- 412 3H4 2---V-------G-SYS---SRM--E------- --WSSY-P- ----------- 413 3 1B3 2GVPARFSGSGSGTSYSLTIGTMEAEDVATYYC QQGSSIPRT FGGGIKLEIKR 414 1C6 2-------------DP--N-HPV-E -I-M-P- --SRKV-W- ----------- 415 2B6 2-----------R-DFT---DPV--D-A----- --NNED-Y- ----------S 416 1G5 2-A-----------DF--N-HPV-ED-I-M-F- --SRKV-Y- ----------- 417 1H6 2---V---------------SS-----A----- --W--Y-L- --A---V-LR- 418 4 1F3 2GVPSRFSGSGSGTSYSLTISSVEAEDAATYYC QQYIGYPYT FGGGTKLEIKR 419 2ES 2---A---------------GAM----V----- --GSSI--- ----------- 420

TABLE 7 Affinities, binding kinetics, and in vitro toxin neutralizationresults of scFv selected from phage antibody libraries scFv K_(d) ^(a)k_(on) k_(off) clone Epitope (M) (10⁴ M⁻¹ s⁻¹) (10⁻³ s⁻¹) ParalysisTime^(b) S25 1 7.3 × 10⁻⁸ 1.1 0.82  85 ± 10^(c) C25 2 1.1 × 10⁻⁹ 30 0.33151 ± 12^(c) C39 2 2.3 × 10⁻⁹ 14 0.32 139 ± 8.9^(c) 1C6 3 2.0 × 10⁻⁸ 132.5  63 ± 3.3 1F3 4 1.2 × 10⁻⁸ 92 11  52 ± 1.4 C25 + S25 Combination 218± 22^(c, d) BoNT/A pure toxin (control)  56 ± 3.8 ^(a)k_(on) and k_(off)were measured by surface plasmon resonance and K_(d) calculated ask_(off)/k_(on). ^(b)Time (min.) to 50% twitch reduction in mousehemidiaphragm assay using 20 nM scFv + 20 pM BoNT/A, compared to timefor BoNT/A alone. For C25 + S25 combination, 20 nM scFv each was used.Each value is the mean ± SEM of at least three observations. ^(c)p <0.01 compared to BoNT/A. ^(d)p < 0.05 compared to C25Discussion.

BoNTs consist of a heavy and a light chain linked by a single disulfidebond. The carboxy-terminal half of the toxin binds to a specificmembrane receptor(s), resulting in internalization, while theamino-terminal half mediates translocation of the toxin from theendosome into the cytosol. The light chain is a zinc endopeptidase whichcleaves an essential synaptosomal protein, leading to failure ofsynaptic transmission and paralysis. Effective immunotherapy mustprevent binding of the toxin to the receptor, since the other two toxinfunctions occur intracellularly. Identification of epitopes on H_(c)which mediate binding is an essential first step, both to the design ofbetter vaccines and to development of a high-titer neutralizingmonoclonal antibody (or antibodies) for passive immunotherapy.

For this work, we attempted to direct the immune response to aneutralizing epitope(s) by immunization with recombinant BoNT/A H_(c).This should lead to the production of antibodies that prevent binding oftoxin to its cellular receptor(s). One limitation of this approach isthe extent to which recombinant H_(c) mimics the conformation of H_(c)in the holotoxin. The fact that 50% of antibodies selected on H_(c)recognize holotoxin suggests significant structural homology for a largeportion of the molecule. Although 50% of antibodies selected on H_(c) donot bind holotoxin, this could result from packing of a significantportion of the H_(c) surface against other toxin domains. Our results donot, however, exclude the possibility that some of these antibodies arebinding H_(c) conformations that do not exist in the holotoxin or thatconformational epitopes present in the holotoxin are absent fromrecombinant H_(c). This could lead to failure to generate antibodies tocertain conformational epitopes. Regardless, immunizing and selectingwith H_(C) resulted in the isolation of a large panel of monoclonalantibodies which bind holotoxin. In contrast, monoclonal antibodiesisolated after immunization with holotoxin or toxoid bind to other toxindomains (H_(N) or light chain) or to nontoxin proteins present in crudetoxin preparations and toxoid (see results from library 1, above, andEmanuel et al. (1996) j. Immunol. Meth., 193: 189-197).

To produce and characterize the greatest number of monoclonal antibodiespossible, we used phage display. This approach makes it possible tocreate and screen millions of different antibodies for binding. Theresulting antibody fragments are already cloned and can easily besequenced to identify the number of unique antibodies. Expression levelsin E. coli are typically adequate to produce milligram quantities ofscFv, which can easily be purified by IMAC after subcloning into avector which attaches a hexahistidine tag to the C terminus. Ultimately,the V_(H) and V_(L) genes can be subcloned to construct complete IcGmolecules, grafted to construct humanized antibodies, or mutated tocreate ultrahigh-affinity antibodies. By this approach, 28 uniquemonoclonal anti-BoNT/A H_(c) antibodies were produced and characterized.

The antibody sequences were diverse, consisting of 3 differentV_(H)-gene families, at least 13 unique V-D-J rearrangements, and 3V_(K)-gene families. Generation of this large panel of BoNT/A H_(c)antibodies was a result of the choice of antigen used for immunizationand selection (BoNT/A H_(c)). For example, a Fab phage antibody libraryconstructed from the V genes of mice immunized with pentavalent toxoidyielded only two Fab which bound pure toxin (in this case, BoNT/B). Themajority of the Fab bound nontoxin proteins present in the toxoid(Emanuel, et al., J. Immunol. Methods 193:189-197 (1996)).

Despite the sequence diversity of the antibodies, epitope mappingrevealed only four nonoverlapping epitopes. Epitopes 1 and 2 wereimmunodominant, being recognized by 21 of 28 (75%) of the antibodies.Interestingly, approximately the same, number (three to five) ofimmunodominant BoNT/A H_(c) peptide (nonconformational) epitopes arerecognized by mouse and human polyclonal antibodies after immunizationwith pentavalent toxoid and by horse polyclonal antibodies afterimmunization with formaldehyde-inactivated BoNT/A (Atassi (1996) J.Protein Chem., 15: 691-699).

scFv binding epitopes 1 and 2 resulted in partial antagonism oftoxin-induced neuroparalysis at the mouse neuromuscular junction. Whenadministered together, the two scFv had an additive effect, with thetime to neuroparalysis increasing significantly. These results areconsistent with the presence of two unique receptor binding sites onBoNT/A H_(c). While the BoNT/A receptor(s) has not been formallyidentified, the results are consistent with those of ligand bindingstudies, which also indicate two classes of receptor binding sites ontoxin, high and low affinity, and have led to a “dual receptor” modelfor toxin binding (Montecucco (1986) Trends Biochem. Sci. 11:314-317).Whether both of these sites are on H_(c), however, is controversial. Intwo studies, BoNT/A H_(c) partially inhibited binding and neuromuscularparalysis (Black, et al. (1986) J. Cell Biol., 103:521-534; Black, etal. (1980) Am. J. Med., 69:567-570), whereas Daniels-Holgate et al.(1996) J. Neurosci. Res. 44:263-271, showed that BoNT/A H_(c) inhibitedbinding at motor nerve terminals but had no antagonistic effect ontoxin-induced neuroparalysis at the mouse neuromuscular junction. Ourresults are consistent with the presence of two “productive” receptorbinding sites on H_(c) which result in toxin internalization andtoxicity. Differences in scFv potency may reflect differences inaffinity of H_(c) for receptor binding sites or may reflect the greaterthan 10-fold difference in affinity of scFv for H_(c). Finally, we havenot formally shown that any of the scFv actually block binding of toxinto the cell surface. It is conceivable that the observed effect on timeto neuroparalysis results from interference with a postbinding event.

ScFv antagonism of toxin-induced neuroparalysis in the mousehemidiaphragm assay was less than that (7.5-fold prolongation of time toneuroparalysis) observed for 2.0×10⁻⁹ M polyclonal equine antitoxin(PerImmune Inc.). This difference could be due to the necessity ofblocking additional binding sites, differences in antibody affinity oravidity, or a cross-linking effect leading to aggregated toxin whichcannot bind. Affinity of antibody binding is also likely to be animportant factor, since the toxin binds with high affinity to itsreceptor (Williams et al. (1983) Eur. J. Biochem., 131: 437-445) and canbe concentrated inside the cell by internalization. Of note, the mostpotent scFv has the highest affinity for H_(c). Availability of otherscFv described here, which recognize the same neutralizing epitope butwith different K_(d)s, should help define the importance of affinity.These scFv, however, differ by many amino acids and may also differ infine specificity, making interpretation of results difficult.Alternatively, mutagenesis combined with phage display can lead to theproduction of scFv which differ by only a few amino acids in sequencebut vary by several orders of magnitude in affinity (Schier et al.(1996) J. Mol. Biol., 263: 551-567). The same approach can be used toincrease antibody affinity into the picomolar range (Id.).

The “gold standard” for neutralization is protection of mice against thelethal effects of toxin coinjected with antibody. While the relationshipbetween in vitro and in vivo protection has not been formallyestablished, equine antitoxin potentially neutralizes toxin in bothtypes of assays (see above and Hatheway et al. (1984) J. Infect. Dis.,150: 407-412). It is believed that this relationship holds for the scFvreported here, and this can be verified experimentally.

Such studies are not possible with small (25-kDa) scFv antibodyfragments. The small size of scFv leads to rapid redistribution (thehalf-life at a phase is 2.4 to 12 min) and clearance (the half-life at βphase is 1.5 to 4 h) and antibody levels which rapidly becomeundetectable (Huston, et al., (1996) J. Nucl. Med. 40: 320; Schier etal. (1995) Immunotechnology, 1: 73-81), while toxin levels presumablyremain high (Hildebrand, et al. (1961) Proc. Soc. Exp. Biol. Med.107-284-289). Performance of in vivo studies will be facilitated by theconstruction of complete IgG molecules from the V_(H) and V_(L) genes ofscFv. Use of human constant regions will yield chimeric antibodies lessimmunogenic than murine monoclonals and much less immunogenic thancurrently used equine antitoxin. Immunogenicity can be further reducedby CDR grafting to yield humanized antibodies.

Example 2 Potent Neutralization of Botulinum Neurotoxin By RecombinantOligoclonal Antibody

The spore-forming bacteria Clostridium botulinum secrete botulinumneurotoxin (BoNT), the most poisonous substance known (Gill (1982)Microbiol. Rev. 46: 86-94). The protein toxin consists of a heavy andlight chain that contain three functional domains (Simpson (1980) J.Pharmacol. Exp. Ther. 212: 16-21; Montecucco and Schiavo (1995) Q. Rev.Biophys. 28: 423-472; Lacy et al. (1998) Nat. Struct. Biol. 5: 898-902).The Cterminal portion of the heavy chain (HC) comprises the bindingdomain, which binds to a sialoganglioside receptor and a putativeprotein receptor on presynaptic neurons, resulting in toxin endocytosis(Dolly et al. (1984) Nature (London) 307: 457-460; Montecucco (1986)Trends Biochem. Sci. 11: 315-317). The N-terminal portion of the heavychain (H_(N)) comprises the translocation domain, which allows the toxinto escape the endosome. The light chain is a zinc endopeptidase thatcleaves different members of the SNARE complex, depending on serotype,resulting in blockade of neuromuscular transmission (Schiavo et al.(1992) Nature (London) 359: 832-835; Schiavo et al. (1993) J. Biol.Chem. 268: 23784-23787).

There are seven BoNT serotypes (A-G; Lacy and Stevens (1999) J. Mol.Biol. 291: 1091-1104), four of which (A, B, E, and F) cause the humandisease botulism (Arnon et al. (2001) J. Am. Med. Assoc. 285:1059-1070). Botulism is characterized by flaccid paralysis, which if notimmediately fatal requires prolonged hospitalization in an intensivecare unit and mechanical ventilation. The potent paralytic ability ofthe toxin has resulted in its use in low doses as a medicine to treat arange of overactive muscle conditions including cervical dystonias,cerebral palsy, posttraumatic brain injury, and poststroke spasticity(Mahant et al. (2000) J. Clin. Neurosci. 7: 389-394). BoNTs are alsoclassified by the Centers for Disease Control (CDC) as one of the sixhighest-risk threat agents for bioterrorism (the “Class A agents”),because of their extreme potency and lethality, ease of production andtransport, and need for prolonged intensive care (Arnon et al. (2001) J.Am. Med. Assoc. 285: 1059-1070). Both Iraq and the former Soviet Unionproduced BoNT for use as weapons (United Nations Security Council (1995)Tenth Report of the Executive Committee of the Special CommissionEstablished by the Secretary-General Pursuant to Paragraph 9(b)(I) ofSecurity Council Resolution 687 (1991), and Paragraph 3 of Resolution699 (1991) on the Activities of the Special Commission (United NationsSecurity Council, New York); Bozheyeva et al. (1999) Former SovietBiological Weapons Facilities in Kazakhstan: Past, Present, and Future(Center for Nonproliferation Studies, Monterey Institute ofInternational Studies, Monterey, Calif.)), and the Japanese cult AumShinrikyo attempted to use BoNT for bioterrorism (Arnon et al. (2001) J.Am. Med. Assoc. 285: 1059-1070). As a result of these threats, specificpharmaceutical agents are needed for prevention and treatment ofintoxication.

No specific small-molecule drugs exist for prevention or treatment ofbotulism, but an investigational pentavalent toxoid is available fromthe CDC (Siegel (1988) J. Clin. Microbiol. 26: 2351-2356) and arecombinant vaccine is under development (Byrne and Smith (2000)Biochimie 82: 955-966). Regardless, mass civilian or militaryvaccination is unlikely because of the rarity of disease or exposure andthe fact that vaccination would prevent subsequent medicinal use ofBoNT. Postexposure vaccination is useless because of the rapid onset ofdisease. Toxin neutralizing antibody (Ab) can be used for pre- orpostexposure prophylaxis or for treatment (Franz et al. (1993) Pp.473-476 in Botulinum and Tetanus Neurotoxins: Neurotransmission andBiomedical Aspects, ed. DasGupta, B. R. Plenum, New York). Smallquantities of both equine antitoxin and human botulinum immune globulinexist and are currently used to treat adult (Black and Gunn (1980) Am.J. Med. 69: 567-570; Hibbs et al. (1996) Clin. Infect. Dis. 23: 337-340)and infant botulism (Arnon (1993) Pp. 477-482 in Botulinum and TetanusNeurotoxins: Neurotransmission and Biomedical Aspects, ed. DasGupta, B.R. Plenum, New York), respectively. Recombinant monoclonal antibody(mAb) could provide an unlimited supply of antitoxin free of infectiousdisease risk and not requiring human donors for plasmapheresis. SuchmAbs must be of high potency to provide an adequate number of doses atreasonable cost. In some instances, the potency of polyclonal Ab can berecapitulated in a single mAb (Lang et al. (1993) J. Immunol. 151:466-472). In the case of BoNT, potent neutralizing mAbs have yet to beproduced: single mAb neutralizing at most 10 to 100 times the 50% lethaldose (LD50) of toxin in mice (Pless et al. (2001) Infect. Immun. 69:570-574; Hallis et al. (1993) Pp. 433-436 In: Botulinum and TetanusNeurotoxins: Neurotransmission and Biomedical Aspects, ed. DasGupta, B.R., Plenum, N.Y.). In this example, we show that BoNT serotype A(BoNT/A) can be very potently neutralized in vitro and in vivo bycombining two or three mAbs, providing a route to drugs for preventingand treating botulism and diseases caused by other pathogens andbiologic threat agents.

Methods

IgG Construction.

V_(H) genes of C25, S25, and 3D12 single-chain fragment variable (scFv)were amplified using PCR from the respective phagemid DNA with theprimer pairs GTC TCC TGA GCT AGC TGA GGA GAC GGT GAC CGT GGT (SEQ IDNO:183) and either GTA CCA ACG CGT GTC TTG TCC CAG GTC CAG CTG CAG GAGTCT (C25, SEQ ID NO:184), GTA CCA ACG CGT GTC TTG TCC CAG GTG AAG CTGCAG CAG TCA (S25, SEQ ID NO:2M 185), or GTA CCA ACG CGT GTC TTG TCC CAGGTG CAG CTG GTG CAG TCT (3D 12, SEQ ID NO:186). DNA was digested withMlu1 and NheI, ligated into N5KG1Val-Lark (gift of Mitch Reff, IDECPharmaceuticals, San Diego) and clones containing the correct V_(H)identified by DNA sequencing. V_genes of C25, S25, and 3D12 scFv wereamplified from the respective phagemid DNA with the primer pairs TCA GTCGTT GCA TGT ACT CCA GGT GCA CGA TGT GAC ATC GAG CTC ACT CAG TCT (SEQ IDNO:187) and CTG GAA ATC AAA CGT ACG TTT TAT TTC CAG CTT GGT (C25, SEQ IDNO:188), TCA GTC GTT GCA TGT ACT CCA GOT GCA CGA TGT GAC ATC GAG CTC ACTCAG TCT (SEQ ID NO:189) and CTG GAA ATC AAA CGT ACG TTT GAT TTC CAG CTTGGT (S25, SEQ ID NO:190), or TCA GTC GTT GCA TGT ACT CCA GGT GCA CGA TGTGAC ATC GTG ATG ACC CAG TCT (SEQ ID NO:191) and CTG GAA ATC AAA CGT ACGTTT TAT CTC CAG CTT GGT (3D 12, SEQ ID NO:192), cloned into pCR-TOPO(Invitrogen) and clones containing the correct V_identified by DNAsequencing. V_genes were excised from pCR-TOPO with DraIII and BsiWI andligated into DraIII- and BsiWI-digested N5KG1Val-Lark DNA containing theappropriate V_(H) gene. Clones containing the correct V_(H) and V_(κ)gene were identified by DNA sequencing, and vector DNA was used totransfect CHO DG44 cells by electroporation. Stable cell lines wereestablished by selection in G418 and expanded into 1 L spinner flasks.Supernatant containing IgG was collected, concentrated byultrafiltration, and purified on Protein G (Pharmacia).

Measurement of IgG Affinity and Binding Kinetics.

IgG binding kinetics were measured using surface plasmon resonance in aBIAcore (Pharmacia Biosensor) and used to calculate the K_(d).Approximately 200-400 response units of purified IgG (10-20 μg/ml in 10mM acetate, pH 3.5-4.5) was coupled to a CM5 sensor chip by usingN-hydroxysuccinimide-N-ethyl-N′-(dimethylaminopropyl)-carbodiimidechemistry. The association rate constant for purified BoNT/AH_(C) wasmeasured under continuous flow of 15 μl/min, using a concentration rangeof 50-800 nM. The association rate constant (k_(on)) was determined froma plot of (1 n(dR/dt))/t vs. concentration. The dissociation rateconstant (k_(off)) was determined from the dissociation part of thesensorgram at the highest concentration of scFv analyzed using a flowrate of 30 μl/min to prevent rebinding. K_(d) was calculated ask_(off)/k_(on).

Measurement of In Vitro Toxin Neutralization.

Phrenic nerve hemidiaphragm preparations were excised from male CD-1mice (25-33 g) and suspended in 135 mM NaCl, 5 mM KCl, 1 mM Na₂HPO₄, 15mM NaHCO₃, 1 mM MgCl₂, 2 mM CaCl₂, and 11 mM glucose. The incubationbath was bubbled with 95% O₂/5% CO₂ and maintained at 36° C. Phrenicnerves were stimulated at 0.05 Hz with square waves of 0.2 ms duration.Isometric twitch tension was measured using a force-displacementtransducer (Model FT03, Grass Instruments, Quincy, Mass.). Purified IgGwere incubated with BoNT A for 30 min at room temperature and then addedto the tissue bath resulting in a final IgG concentration of 6.0×10⁻⁸ M(S25 and 3D12 alone) or 2.0×10⁻⁸ M (C25 alone) and a final BoNT Aconcentration of 2.0×10⁻¹¹ M. For pairs of IgG, the final concentrationof each IgG was decreased 50%, and for studies of a mixture of all 3IgG, the concentration of each IgG was decreased by 67%.

Measurement of In Vivo Toxin Neutralization.

Fifty micrograms of the appropriate IgG were added to the indicatednumber of mouse LD₅₀ of BoNT/A neurotoxin (Hall strain) in a totalvolume of 0.5 ml of gelatin phosphate buffer and incubated at RT for 30min. For pairs of Ab, 25 μg of each Ab was added, and for thecombination of 3 Ab, 16.7 μg of each Ab was added. The mixture was theninjected i.p. into female CD-1 mice (16-22 g). Mice were studied ingroups of ten and were observed at least daily. The final death tallywas determined 5 days after injection.

Measurement of Solution Affinity of mAbs.

Equilibrium binding studies were conducted using a KinExA flowfluorimeter to quantify the antibodies with unoccupied binding sites inreaction mixtures of the antibody with the antigen. Studies withreaction mixtures comprised of one, two, or three different antibodieswere conducted in Hepes-buffered saline, pH 7.4, with total antibodyconcentrations of 342, 17.2, and 17.2 pM, respectively. In all cases,the concentration of soluble toxin was varied from less than 0.1 togreater than 10-fold the value of the apparent K_(d) (twelveconcentrations, minimum). Reaction mixtures comprised of one, two, orthree different antibodies were incubated at 25° C. for 0.5, 3, and 17h, respectively, to ensure that equilibrium was achieved.

Results

To generate mAbs capable of neutralizing BoNT/A, we previously generatedscFv phage antibody libraries from mice immunized with recombinantBoNT/A binding domain (H_(C)) and from humans immunized with pentavalentbotulinum toxoid (Amersdorfer et al. (1997) Infect. Immun. 65:3743-3752; Amersdorfer et al. (2002) Vaccine 20: 1640-1648). Afterscreening more than 100 unique mAbs from these libraries, three groupsof scFv were identified that bound nonoverlapping epitopes onBoNT/AH_(C) and that neutralized toxin in vitro (prolonged the time toneuroparalysis in a murine hemidiaphragm model; Amersdorfer et al.(1997) Infect. Immun. 65: 3743-3752; Amersdorfer et al. (2002) Vaccine20: 1640-1648). In vitro toxin neutralization increased significantlywhen two scFv binding nonoverlapping epitopes were combined. In vivotoxin neutralization could not be determined because of the rapidclearance of the 25-kDa scFv from serum (Colcher et al. (1990) J. Natl.Cancer Inst. 82: 1191-1197).

To evaluate in vivo BoNT neurotoxin neutralization, IgG were constructedfrom the VH and V_genes of three BoNT/A scFv that neutralized toxin invitro. V_(H) and Vκ genes were sequentially cloned into a mammalianexpression vector, resulting in the fusion of the human Cκ gene to theVκ and the human γ1 gene to the V_(H). Stable expressing cell lines wereestablished and IgG purified from supernatant yielding chimeric IgG withmurine V-domains and human C-domains, for the murine scFv C25 and S25,and a fully human IgG for the human scFv 3D12. IgG equilibrium bindingconstants (K_(d)) were measured and found to be at least comparable tothe binding constants of the scFv from which they were derived (Table8). The antigen binding affinity of two of the IgG (S25 and 3D12) wassignificantly higher (lower K_(d)) than for the corresponding scFv,largely because of an increase in the association rate constant(k_(on)). We presume this reflects an increase in the stability of themolecule and hence an increase in the functional antibody concentration.

TABLE 8 Association (k_(on)) and dissociation (k_(off)) rate constantsand equilibrium dissociation constants (K_(d)) for BoNT/A IgG and scFvfrom which the IgG were derived. IgG scFv Ab K_(d) k_(on) k_(off) K_(d)k_(on) k_(off) C25 1.69 × 10⁻⁹ 1.32 × 10⁶ 2.24 × 10⁻³ 1.10 × 10−9 3.00 ×10⁵ 3.30 × 10⁻⁴ S25 3.90 × 10⁻⁹ 1.46 × 10⁶ 5.70 × 10⁻³ 7.30 × 10−8 1.10× 10⁴ 8.10 × 10⁻⁴ 3D12 5.62 × 10⁻¹¹ 2.26 × 10⁶ 1.27 × 10⁻⁴ 3.69 × 10−81.30 × 10⁴ 5.00 × 10⁻⁴

In vitro toxin neutralization by IgG was determined in the mousehemidiaphragm assay (Desphande et al. (1995) Toxicon 33: 551-557).Compared with toxin alone, each of the three IgG significantly increasedthe time to neuroparalysis, with C25 being the most potent (FIG. 4).Significant synergy in toxin neutralization was observed when pairs ofIgG were studied. For these studies, it was necessary to decrease theconcentration of C25 IgG studied 3-fold to 20 nM because of its highpotency and the fact that the hemidiaphragm preparations have an 8-hlifespan. Each pair of IgG significantly increased the time toneuroparalysis compared with the time for either single IgG (FIG. 4). Amixture of all three IgG further increased the time to neuroparalysis,although this difference did not reach statistical significance comparedwith antibody pairs because of the small number of diaphragms studied.

In vivo toxin neutralization was studied using a mouse assay in whichtoxin and Ab are premixed and injected i.p., and time to death andnumber of surviving mice determined (Sheridan et al. (2001) Toxicon 39:651-657). Fifty micrograms of each single mAb prolonged the time todeath but failed to protect mice challenged with 20 LD₅₀s (FIG. 5A). Incontrast, any pair of mAbs completely protected mice challenged with 100LD₅₀s of toxin (FIG. 5B). At 500 LD₅₀s, the majority of mice receivingtwo of the pairs of mAbs (S25+3D12 or C25+S25) died, whereas 80% of micereceiving the pair of C25+3D12 survived (FIG. 3). All mice receiving amixture of all three mAbs (oligoclonal Ab) survived challenge with 500LD₅₀s of toxin (FIG. 6). In these studies, the total amount of Abadministered was kept constant at 50 μg per mouse. To determine potency,mAb pairs and oligoclonal Ab were studied at increasing doses of toxin(FIG. 6). The most potent mAb pair (C25+3D12) protected 90% of micechallenged with 1,000 LD₅₀s, with no mice surviving challenge with 2,500LD₅₀s. In contrast, oligoclonal Ab completely protected all micechallenged with 5,000 LD₅₀s of toxin, with five of ten mice survivingchallenge with 20,000 LD₅₀s of toxin. The potency of the oligoclonal Abwas titrated using a modification of the standard mouse neutralizationbioassay (Hatheway and Dang (1994) Pp. 93-107 In: Therapy with BotulinumToxin, ed. Jankovic, J., Dekker, N.Y.) and was determined to be 45international units (IU)/mg of Ab, 90 times more potent than the humanbotulinum immune globulin used to treat infant botulism (Arnon (1993)Pp. 477-482 in Botulinum and Tetanus Neurotoxins: Neurotransmission andBiomedical Aspects, ed. DasGupta, B. R. Plenum, N.Y.). By definition,one IU neutralizes 10,000 LD50s of BoNT/A toxin (Bowmer (1963) Bull.W.H.O. 29: 701-709).

Two potential mechanisms could account for the increase in potencyobserved when mAbs were combined: an increase in the functional bindingaffinity of the Ab mixture for toxin and/or an increase in the blockadeof the toxin surface that binds to cellular receptor(s). To determinethe effect of combining antibodies on the functional binding affinities,apparent K_(d) were determined for each single mAb, pairs of mAbs, andthe mixture of all three mAbs by using a flow fluorimeter to quantifythe free antibody that remained in solution reaction mixtures. Forsingle mAbs, the antigen binding affinities measured in homogeneoussolution (both antigen and antibody in solution; FIG. 7) were lower(higher K_(d)) than those measured by surface plasmon resonance in aBIAcore (Table 8), where the antibody is immobilized and only theantigen is in solution. When antibody C25, which showed the greatest invitro potency, was mixed in equimolar amounts with antibody 3D12, theresulting Ab combination bound to the toxin with an apparent K_(d) of 65pM, an affinity 200- and 10-fold higher (lower K_(d)) than thoseobserved with the individual antibodies alone. Addition of equimolaramounts of a third mAb (S25) to the mixture increased the apparentaffinity further to 18 pM. An equimolar mixture of C25 with S25 yieldedonly a minor 2-fold increase in affinity, which may explain why thispair is less potent in vivo than the combination of C25 and 3D12. Theincrease in functional affinity observed with multiple mAbs may be dueto either a conformational change in toxin that occurs on binding of thefirst mAb, resulting in higher affinity binding of the second and thirdmAbs, or from mAb binding changing the toxin from a monovalent to amultivalent antigen (Moyle et al. (1983) J. Immunol. 131: 1900-1905).This results in an “avidity effect” and an increase in affinity. Avidityeffects have been well recognized and characterized for IgG binding tomultivalent antigens (Crothers and Metzger (1972) Immunochemistry 9:341-357), such as cell surfaces, but are not well appreciated asoccurring in solution.

The increments in measured Kd are consistent with the increase in invivo potency observed for mAb pairs and oligoclonal Ab. Rearranging theequilibrium binding equation:free toxin/bound toxin=K_(d)/[serum antibody].Assuming a 2-ml mouse blood volume, the serum antibody concentration is160 nM when mice receive 50 μg of Ab.

Because the administered amount of toxin is a large multiple of theLD₅₀, bound toxin˜administered toxin. Thus, the above equationsimplifies to:free toxin/administered toxin=K_(d)/160 nM.To determine the amount of administered toxin that results in death of50% of mice, one substitutes 1 LD₅₀ for the amount of free toxin andsolves for administered toxin, yielding the equation:administered toxin (in LD ₅₀ s)=1 LD ₅₀×160 nM/K_(d).Using the solution Kd for C25, the predicted toxin dose at which 50% ofthe mice survive is 16 LD₅₀s (administered toxin=1 LD₅₀×160 nM/10 nM).When this calculation is applied to the C25 and 3D12 Ab pair, and tooligoclonal Ab, the magnitude of the increase in potency on combiningantibodies parallels the increase in functional affinity (Table 9).

TABLE 9 Observed and predicted toxin neutralization by rewcombinantantibody. Predicted Toxin Observed Toxin Antibody NeutralizationNeutralization C25   16 LD₅₀s   <20 LD₅₀s C25 + 3D12 2,500 LD₅₀s  1,500LD₅₀s C25 + 3D12 + S25 8,900 LD₅₀s 20,000 LD₅₀s

The second potential mechanism for potent toxin neutralization byoligoclonal Ab is the need to block multiple epitopes on the toxinbinding domain surface that bind to cellular receptors. It has beenhypothesized that the toxin binds to cellular receptors via at least twosites on the toxin binding domain (. Dolly et al. (1984) Nature (London)307: 457-460; Montecucco (1986) Trends Biochem. Sci. 11: 315-317). Theseinclude a ganglioside binding site and a putative protein receptorbinding site. In fact, two spatially separated ganglioside binding siteshave been observed in the co-crystal structure of the homologous tetanustoxin (Fotinou et al. (2001) J. Biol. Chem. 276: 32274-32281), and mAbsbinding nonoverlapping tetanus toxin epitopes can block binding of toxinto GT1b ganglioside (Fitzsimmons et al. (2000) Vaccine 19: 114-121). Ourprior epitope mapping studies are consistent with multiple mAbs blockinga large portion of the BoNT binding domain (HC) (Mullaney et al. (2001)Infect. Immun. 69: 6511-6514). Two of the mAbs (S25 and 3D12) bind theC-terminal subdomain of BoNT HC. The C25 mAb binds a conformationalepitope that consists of sequence from the N- and C-terminal subdomainsof BoNT H_(C). One model consistent with the epitope mapping places thethree mAb epitopes on the same H_(C) face and overlapping the knowndocking sites for the putative cellular ganglioside receptor GT1b(Mullaney et al. (2001) Infect. Immun. 69: 6511-6514).

Discussion

In conclusion, we have shown that one of the six class A biowarfareagents, BoNT/A, can be potently neutralized by an oligoclonal Abconsisting of only three mAbs. Oligoclonal Ab is 90 times more potentthan hyperimmune human globulin and approaches the potency ofhyperimmune mono-serotype horse type A antitoxin (Sheridan et al. (2001)Toxicon 39: 651-657). Thus, the potency of polyclonal serum can bedeconvoluted, or reduced, to mAbs binding only three nonoverlappingepitopes. This synergistic effect results in a more than 20,000-foldincrease in potency for the three mAbs compared with the potency of anyof the single mAbs. Others have previously shown synergy betweenmonoclonal antibodies in neutralizing tetanus toxin or HIV infection. Inthe case of tetanus toxin, combining three to four monoclonal antibodiesincreased the potency of in vivo toxin neutralization up to 200-fold(Volk et al. (1984) Infect. Immun. 45: 604-609). In the case of HIV,combining three or four mAbs increased the potency of viralneutralization 10-fold compared with individual mAbs (Zwick et al.(2001) J. Virol. 75: 12198-12208). Thus, our observation is likely toprove general in many systems. We show, however, that the increasedpotency in the case of toxin neutralization likely results from a largeincrease in the functional affinity of the mixture antibodies. Whethersuch a mechanism holds true for viral neutralization is unclear.

One can hypothesize that the polyclonal humoral immune response to toxinis functionally dominated by Ab binding only a few nonoverlappingepitopes. The increase in potency appears to result primarily from alarge decrease in the K_(d) of oligoclonal Ab compared with theindividual mAb, and also to greater blockade of the toxin surface thatinteracts with cellular receptors Such mechanisms may be generallyapplicable to many antigens in solution, suggesting that oligoclonal Abmay offer a general route to more potent antigen neutralization thanmAb. Although it might be possible to achieve a similar potency byengineering the K_(d) of the C25 mAb to near pM, oligoclonal Ab offers asimpler, more rapid route to a potent antitoxin.

Oligoclonal Ab also offers a safe and unlimited supply of drug forprevention and treatment of BoNT/A intoxication. Because the Ab consistsof either chimeric or human IgG, production could be immediately scaledto produce a stockpile of safe antitoxin. Alternatively, we have alreadyreplaced the chimeric S25 IgG with a fully human IgG and increasedpotency of the oligoclonal Ab more than 2-fold. Work is ongoing toreplace chimeric C25 with a fully human homologue. Chimeric, humanized,and human mAb represent an increasingly important class of therapeuticagents whose means of production are known. Ten mAbs have been approvedby the FDA for human therapy and more then 70 other mAb therapeutics arein clinical trials (Reichert (2001) Nat. Biotechnol. 19: 819-822). Withan elimination half-life of up to 4 weeks, Ab could provide months ofprotection against toxin or be used for treatment. Oligoclonal Ab wouldbe applicable to the other BoNT toxin serotypes, as well as to otherclass A agents. Anthrax is a toxin-mediated disease, and Ab has beenshown to be protective for this agent (Little et al. (1997) Infect.Immun. 65: 5171-5175; Beedham et al. (2001) Vaccine 19: 4409-4416).Vaccinia immune globulin can be used to prevent or treat smallpox orcomplications arising from vaccination of immunocompromised hosts (Feery(1976) Vox Sang. 31: 68-76). Ab may also be useful for plague anddisease caused by the hemorrhagic fever viruses (Hill et al. (1997)Infect. immun. 65: 4476-4482; Wilson et al. (2000) Science 287:1664-1666). Our data support the rapid development and evaluation ofoligoclonal Ab for countering BoNT and other agents of biowarfare andbioterrorism.

Example 3 Genetic and Immunological Comparison of Anti-Botulinum Type AAntibodies from Immune And Non-Immune Human Phage Libraries

Understanding the antibody response in botulinum intoxication isimportant for vaccine design and passive prophylaxis. To investigatethis activity, we have studied the immune response to BoNT/A (botulinumneurotoxin serotype A) binding domain (H_(C)) at the molecular levelusing phage display. The scFv antibodies were isolated from V-generepertoires prepared from (a) human volunteer immunized with pentavalentbotulinum toxoid and (b) non-immune human peripheral blood lymphocytesand spleenocytes. A large panel of serotype specific phage expressingbotulinum binding scFv could be selected from both libraries. Epitopemapping of immune scFv binders towards BoNT/A HC revealed surprisingly alimited number of scFv recognizing conformational epitopes thatcorresponded to two distinct groups, clusters I and II. Only scFv fromcluster I exhibited neutralizing activity in the mouse hemidiaphragmassay. Anti-BoNT/A HC clones derived from a non-immune library could beconveniently grouped into clusters III-XI and appeared to share nooverlapping epitopes with cluster I or II. In addition they showed noneutralization of toxin at biologically significant concentrations. Wetherefore suggest that a vaccine based on the pentavalent botulinumtoxoid directs the humoral immune response to a limited number ofimmunodominant epitopes exposed on the binding domain HC.

INTRODUCTION

Botulinum toxin is a paralytic neurotoxin existing as seven differentserotypes (A-G) elaborated by a number of bacterial species belonging tothe genus Clostridium (Hatheway (1989) Pp. 3-24 In: Simpson L L, editor.Botulinum neurotoxin and tetanus toxin. San Diego: Academic Press). Theyare produced as a single chain protein (Mr: 150,000) and fully activatedby limited proteolysis, which results in formation of two chains, theheavy (M_(r): 100,000) and light (Mr: 50,000) chains held together by adisulfide bond and non-covalent bonds (Niemann (1991) Pp. 303-348 In:Alouf J E, Freer J H, editors. Sourcebook of bacterial protein toxins.New York: Academic Press; Simpson (1990) J Physiol., 84:143-151).Poisoning can occur by ingestion of clostridia-contaminated food(foodborne botulism), by infant bowel infection (infant botulism), andby deep subcutaneous infection of wounds (wound botulism). Humanbotulism is most frequently caused by types A, B, and E and rarely by F(Dowell (1984) Rev Infect Dis., 6 (Suppl 1):202-207; Botulism in theUnited States. Handbook for epidemiologists, clinicians and laboratoryworkers. Atlanta, Center for Disease Control, 1980). BoNTs (botulinumneurotoxin serotypes) act preferentially on cholinergic nerve endings toblock acetylcholine release (Habermann et al. (1986) Curr Top MicrobiolImmunol., 129:93-179; Montecucco et al. (1994) Mol Microbiol., 13:1-8).The action of BoNTs involves three steps (Simpson (1986) Ann RevPharmacol Toxicol., 26:427-453): (1) binding to receptors on thepresynaptic membranes via the C-terminus of the heavy chain HC; (2)translocation of the light chain into the cytosol via the N-terminus ofthe heavy chain HN; and (3) cleavage of one or more key components inthe synaptic vesicle docking and fusion protein complex by the zincprotease activity of the light chain (Montecucco et al. (1994) MolMicrobiol., 13:1-8; Schiavo (1992) J Biol Chem., 267:23479-23483;Schiavo et al. (1995) Curr Top Microbiol Immun., 195:257-275). Passiveimmunotherapy has been established as a valuable prophylactic andtherapeutic approach against human pathogens and their toxins (forreview, see Gronski et al. (1990) Mol Immunol., 28:1321-1332 and Cross(1997) P. 97 In: Cryz S J, editor. Immunotherapy and vaccines. Weinheim,Germany: VCH Verlagsgesellschaft). In the case of botulism it isbelieved that antibody preparations recognizing the C-terminal domain ofthe BoNT heavy chain (HC) are able to prevent binding of the toxin toits cellular receptor(s). Immunization of mice with recombinant HCconferred good protection in vivo to a challenge dose up to 1,000,000mouse i.p. LD₅₀ (Clayton et al. (1995) Infect Immun., 63:2738-2742;Byrne et al. (1998) Infect Immun., 66: 10). Equine plasma-derivedpolyclonal anti-botulinum antibody preparations (equine HIG) have beenadministered to more than 80% of adult botulism patients in the past(Middlebrook and Brown (1995) Curr Top Microbiol Immun., 195:89-122;Tacket et al. (1984) Am J Med., 76:794-798; Morris (1981) P. 15 In:Lewis G E jr, editor. Biomedical aspects of botulism. New York: AcademicPress). The large number of different epitopes recognized by polyclonalantibody preparations normally ensures the presence of protectiveantibodies, which are usually a small subpopulation of the totalantibody. For prophylaxis, equine antibody is most effective whenadministered prior to exposure, but can prevent the disease up to 24 hpost exposure (Middlebrook and Brown (1995) Curr Top Microbiol Immun.,195:89-122). However, administration of equine antitoxin can causeadverse reactions, such as serum sickness and anaphylaxis in 9% of cases(Black and Gunn (1980) Am J Med., 69:567-570). Recent efforts have beenfocused on the production of human immunoglobulin (human BIG) preparedfrom serum of immunized volunteer donors (Arnon (1993) Pp. 477-482 In:DasGupta B R, editor. Botulinum and tetanus neurotoxins,neurotransmission and biomedical aspects. New York: Plenum Press).Neutralizing monoclonal antibodies, especially if of human origin, wouldprovide an unlimited source of antibody and replace the preparation ofantibody from humans or horses.

We have been using antibody phage display to generate monoclonalantibodies capable of neutralizing BoNTs (Hoogenboom et al. (1991) NuclAcids Res., 19:4133-4137; McCafferty et al. (1990) Nature, 348:552-554;Skerra and Pluckthun (1988) Science, 240:1038-1041). Using phageantibody libraries constructed from immunized mice, we identified twosets of monoclonal which bound two non-overlapping neutralizing epitopeson BoNT/A HC (Amersdorfer et al. (1997) Infect. Immun., 65:3743-3752).In the present example, we describe the characterization of monoclonalantibodies selected from a phage antibody library constructed from ahuman volunteer immunized with pentavalent botulinum toxoid (A-E). Theaffinities and epitopes recognized by these monoclonal antibodies werecompared to affinities and epitopes recognized by monoclonal antibodiesselected from a non-immune human phage library. The results identify anadditional neutralizing epitope and provide a path to generating a fullyhuman antibody for botulism prevention and treatment.

Materials and Methods:

Immune and Non-Immune V-Gene Antibody Libraries

For construction of an immune phage antibody library, a human volunteerreceived immunization with pentavalent botulinum toxoid types A-E(Michigan Department of Public Health). The volunteer was immunized at0, 2 and 12 weeks with 0.5 ml of pentavalent toxoid and boosted with 0.5ml of toxoid 1 year later. The neutralization titer against BoNT/A wasmeasured using the mouse serum neutralization bioassay (Hatheway et al.(1984) J Infect Dis., 150: 407-412). PBLs were isolated bycentrifugation in Histopaque 1077 and RNA prepared using a modifiedmethod of Cathala et al. (Cathala et al. (1983) DNA, 2:329-335). Firststrand cDNA was made from RNA prepared from 1.0×10⁸ B cells, using anIgG constant region primer for heavy chain or κ and λ constant regionprimers for light chains [26]. VH, Vκ and Vλ genes were amplified fromfirst strand cDNA as described (Marks et al. (1991) J Mol Biol.,222:581-597). PCR products were gel purified, ethanol precipitated afterextraction from the gel and used to construct scFv gene repertoires aspreviously described (Id.). The scFv gene repertoires were gel purifiedand then used as template for re-amplification with flankingoligonucleotides containing appended restriction sites (Id.). scFv generepertoires (VH-Vκ, VH-Vλ) were gel purified, digested with SfiI andNotI, extracted with phenol/chloroform, and ligated into the vectorpCANTAB-5E (Pharmacia Biotech, Milwaukee, Wis.) digested with SfiI andNotI (Sambrook et al. (1991) New York: Cold Spring Harbor Laboratory).The ligation mix was extracted with phenol/chloroform, ethanolprecipitated, and electroporated into 50 μl E. coli TG1 cells (Gibson(1984) University of Cambridge: studies on the Epstein-Barr virusgenome). Cells were plated on TYE plates containing 100 g/ml ampicillinand 1% (w/v) glucose. Colonies were scraped off the plates into 2 ml 2×TY containing 100 μg/ml ampicillin, 1% (w/v) glucose and 15% (v/v)glycerol for storage at −70° C. The products from four transformationsresulted in a library of 7.7×10⁵ individual recombinants. For thenon-immune library, a previously reported phage-displayed human singlechain antibody library containing 6.7×10⁹ members was utilized (Sheetset al. (1997) Proc Natl. Acad Sci USA, 95:6157-6162).

Phage Preparation and Selections

Phagemid particles from both libraries were prepared by rescue withVCS-M13 helper phage (Stratagene) as previously described (Marks et al.(1991) J Mol Biol., 222:581-597). Phage particles were purified andconcentrated by two PEG precipitations (Sambrook et al. (1991) New York:Cold Spring Harbor Laboratory), resuspended in 2 ml phosphate-bufferedsaline (PBS: 25 mM NaH2PO4, 125 mM NaCl, pH 7.4) and filtered through a0.45 μm filter (Nalgene) to achieve a titer of approximately 10¹³transducing units (TU)/ml.

Libraries were selected using 75 mm×12 mm immunotubes (Nunc, Maxisorb)coated overnight at 4° C. with 2 ml of BoNT serotypes A, B, C, and E (50μg/ml each), or BoNT/A HC (50 μg/ml) in PBS, pH 7.4 (Emanuel et al.(1996) J Immunol Meth., 193:189-97). Tubes were blocked with 2% skimmedmilk powder in PBS for 1 h at RT, and then the selection, washing andelution procedures were performed as previously described (Marks et al.(1991) J Mol Biol., 222:581-597) using phage at a concentration of5.0×10¹² TU/ml. The 500 μl of the eluted phage were used to infect 10 mllog phase growing E. coli TG1, which were plated on 2×TY-AMP-Glu plates.Phage were rescued, concentrated as described above, and used for thenext selection round. The rescue-selection-plating cycle was typicallyrepeated for four rounds.

ELISA Screening and Fingerprinting

After each round of selection, single ampicillin-resistant colonies wereused to inoculate microtitre plate wells containing 150 μl of2×TY-AMP-0.1% glucose. The bacteria were grown to give an A600 ofapproximately 0.9, and scFv expression induced by addition ofisopropyl-β-d-thiogalacto-pyranoside (IPTG) to a final concentration of1 mM (De Bellis and Schwartz (1990) Nucl Acids Res., 18:1311). Bacteriawere grown overnight with shaking at 25° C., the cells were pelleted bycentrifugation, and the supernatant containing soluble scFv wascollected. Screening of scFv for binding to BoNTs and BoNT/A HC wasperformed in 96-well microtitre plates (Falcon 3912) coated with 10μg/ml of antigen in PBS, pH 7.4. The scFv derived from the non-immunelibrary were detected using mouse monoclonal antibody 9E10 (1 μg/ml)(Santa Cruz Biotechnology, CA), which recognizes the C-terminal myc tag(Munro and Pelham (1986) Cell, 46:291-300) followed byperoxidase-conjugated anti-mouse Fc antibody (Sigma) as described(Griffiths and Malmqvist (1993) EMBO J., 12:725-734). The scFvs derivedfrom the immune library were detected using peroxidase-conjugatedmonoclonal antibody anti-E (2.5 μg/ml) (Pharmacia Biotech). The reactionwas stopped after 30 min with NaF (3.2 mg/ml) and A405 nm was measured.The number of unique clones was determined by PCR-fingerprinting (Markset al. (1991) J Mol Biol., 222:581-597) followed by DNA sequencing ofthe V_(H) and V_(L) genes of at least two clones from each fingerprintpattern. The specificity of antibodies was determined by ELISA performedas above using wells coated with 10 μg/ml of BoNT/A, BoNT/B, BoNT/C,BoNT/E, BoNT/A H_(C) and recombinant translocation domain of serotype A(BoNT/A HN). Clones were identified as being specific for the selectedantigen if they gave at least a five-fold higher signal than background.

Subcloning, Expression and Purification of scFv

scFv antibodies binding BoNT/A and BoNT/A HC as determined by ELISA weresubcloned into the expression vector pUC119 Sfi-NotmycHis, resulting inthe fusion of a hexa-histidine tag at the C-terminus of the scFv (Schieret al. (1995) Immunotech., 1:73-81). The scFv was expressed and purifiedby immobilized metal affinity chromatography as previously described(Schier et al. (1996) J Mol Biol., 255:28-43) and the concentration ofpurified monomeric scFv determined spectrophotometrically, assuming anA280 nm of 1.0 correlates to an scFv concentration of 0.7 mg/ml.

Epitope Mapping and Affinity Determination

Epitope mapping and kinetic studies were performed using surface plasmonresonance in a BIAcore (Pharmacia Biosensor). In a BIAcore flow cell,approximately 600 resonance units (RU) of BoNT/A HC (15 μg/ml in 10 mMsodium acetate, pH 4.5) were coupled to a CM5 sensor chip using NHS-EDCchemistry (Johnson et al. (1991) Anal Biochem., 198:268-277). Thisamount of coupled BoNT/A HC resulted in scFv RUmax of 100-175 RU. Thesurface was regenerated after binding of scFv using 4 M MgCl₂. Forepitope mapping studies, the amount (RU) of scFv bound for each memberof a pair was determined, and then the two scFv were mixed together togive a final concentration equal to the concentration used formeasurements of the individual scFv (Amersdorfer et al. (1997) Infect.Immun., 65:3743-3752). The Kd of scFv was calculated from theassociation rate constants (k_(on)) and dissociation rate constants(k_(off)) determined in the BIAcore (K_(d)=k_(off)/k_(on)). Associationwas measured under continuous flow of 5 μl/min using a concentrationrange of scFv from 50 to 1000 nM. The k_(on) was determined from a plotof 1 n (dR/dt)/t versus concentration (Karlsson et al. (1991) J ImmunolMeth., 145:229-240). The k_(off) was determined from the dissociationpart of the sensorgram at the highest concentration of scFv analyzedusing a flow rate of 30 μl/min to prevent rebinding.

In Vitro Bioassay

In vitro neutralization studies were performed using a mousehemidiaphragm preparation, as previously described (Desphande (1995)Toxicon, 33:551-557). Phrenic nerve-hemidiaphragm preparations wereexcised from male CD/1 mice (25-33 g) and suspended in 135 mM NaCl, 5 mMKCl, 1 mM Na₂PO₄ 15 mM NaHCO₃ 1 mM MgCl₂ 2 mM CaCl₂, and 11 mM glucose.The incubation bath was bubbled with 95% O2, 5% CO and maintained at 36°C. Phrenic nerves were stimulated at 0.05 Hz with square waves of 0.2 msduration. Isometric twitch tension was measured using aforce-displacement transducer (Model FT03, Grass) connected to a chartrecorder. Purified scFv antibodies were incubated with BoNT/A for 30 minat RT and then added to the tissue bath resulting in a final scFvconcentration of 2.0×10⁻⁸ M and a final BoNT/A concentration of2.0×10⁻¹¹ M. Toxin induced paralysis was defined as a 50% reduction ofthe initial muscle twitch. The ratio of prolongation was calculated fromthe value of 50% reduction by the antibody divided by 50% reduction ofBoNT/A. The combination of 3D12 and C25 was studied at a finalconcentration of 2.0×10⁻⁸ M each. Differences between times to 50%twitch reduction were determined using two-tailed t-test, with P<0.05being significant.

Preparation of Botulinum Toxin and Botulinum Toxin Domains

Purified botulinum toxin serotype A, B, C and E (150 kDa) were obtainedfrom USAMRIID. The binding domain of botulinum toxin type A (BoNT/A HC)was expressed in E. coli and purified by immobilized metal affinitychromatography (IMAC) utilizing a C-terminal (His₆) tag (OphidianPharmaceuticals, Inc.). The translocation domain of botulinum toxin typeA (BoNT/A HN) was a gift from Dr. R. Stevens (UC-Berkeley, CA).

TABLE 10 Specificity of BoNT binding scFv selected from immune andnon-immune phage display libraries Number of unique scFv Immune LibraryscFv specificity (pentavalent toxoid) Non-Immune Library BoNT/A 23 14 HC(binding domain) 6 10 HN (translocation domain) 4 1 Light chain (cat.domain) 13 3 BoNT/B 16 5 BoNT/C 6 5 BoNT/E 3 3Results

Strategy for the Synthesis of Immune Phase Display Library

PBLs from a human volunteer immunized with pentavalent botulinum toxoidwere used to generate a scFv phage antibody library. The donorspolyclonal serum was protective against BoNT/A with a titer of 2.56 IU(international units) in the mouse neutralization bioassay (Hatheway etal. (1984) J Infect Dis., 150: 407-412). The V_(H) and V_(L) genes wereamplified from RNA, spliced together to create scFv gene repertoires andcloned into pCANTAB-5E to create a phage antibody library of 7.7×10⁵transformants. PCR screening of 15 randomly selected clones indicatedthat all carried full length inserts, 66% having Vκ light chains and 34%having V_(λ) light chains as determined by germline gene specific lightchain primers (data not shown).

Selection of Phase Antibody Libraries and ELISA Screening

Both the immune library and a large non-immune human phage antibodylibrary (Sheets et al. (1997) Proc Natl. Acad Sci USA, 95:6157-6162)were selected on BoNT serotypes A, B, C, E and BoNT/A HC. After threerounds of selection on BoNT/A or BoNT/A HC, the frequency of ELISApositive clones was 79 and 100%, respectively from the immune library. Asimilar frequency of ELISA positivity was observed for the otherserotypes. After three rounds of selection on BoNT/A or BoNT/A HC, thefrequency of ELISA positive clones was 28 and 94%, respectively from thenon-immune library. A similar frequency of ELISA positivity was observedfor the other serotypes. The number of unique scFv was determined by DNAfingerprinting followed by DNA sequencing, and specificity of each scFvwas determined by ELISA. In screening, 100 colonies from each selection,48 unique antibodies were identified from the immune library (23 BoNT/A,16 BoNT/B, 6 BoNT/C and 3 BoNT/E) and 27 unique antibodies from thenon-immune library (14 BoNT/A, 5 BoNT/B, 5 BoNT/C and 3 BoNT/E) (Table10).

The fine specificity of each BoNT/A scFv was determined by ELISA onrecombinant BoNT/A HC and BoNT/A HN domains (FIG. 8). Of the 23 immuneBoNT/A antibodies isolated after selection on toxin, 6 bound to BoNT/AHC (3A6, 3D12, 2A1, 3B8, 3F10, 2B11), 4 bound to BoNT/A HN (3D4, 3A11,4A4, 3G4) (FIG. 8A) and the remaining 13 antibodies presumably bound thelight chain (Chen et al. (1997) Infect Immun., 65:1626-1630). Thesefindings suggest that immunization with botulinum toxoid directs theimmune response towards the light chain, with fewer antibodies directedagainst the HC or HN domains.

Selection of the immune library on BoNT/A HC yielded only a singleunique antibody (2A1), which was clonally related to toxin selectedclones 3D12 and 3D6 (Table 11). When the VL gene usage of the sixanti-HC clones was analyzed, all were found to use the Vκ1 gene family(Table 11), although the library contained ⅔ Vκ and ⅓ Vλ light chaingenes. Selection of the non-immune library on BoNT/A holotoxin yieldedfour antibodies, but none of these bound BoNT/A HC. Selection of thelibrary on BoNT/A HC yielded 10 unique scFv, which used both Vκ or Vλlight chain genes (Table 11). Overall, only 50% of these scFv boundholotoxin, consistent with the observation that a significant portion ofthe HC surface is buried in the holotoxin (Lacy et al. (1998) Nat StructBiol., 5:898-902). All scFv antibodies were serospecific and domainspecific, with no cross reactivity observed except for clone 2B11 fromthe non-immune library, which bound to BoNT/A HC and BoNT/A HN domain asdetermined by ELISA (FIG. 8B).

TABLE 11 CDR 3-sequences and affinities for human scFv antibodiesisolated from immune and non-immune libraries, selected on BoNT/A andBoNT/A H_(c).^(a) Non-immune library Heavy Chain Diff from Clone FamilySegment Genome V_(H) CDR3 2A9^(b) V3 DP54 5 GRGVN (SEQ ID NO:193)2B1^(b) V3 DP46 0 NGDPEAFDY (SEQ ID NO:194) 2H6^(b) V3 DP47 6 ALQSDSPYFD(SEQ ID NO:195) 3C2^(b) V3 DP46 2 DLAIFAGNDY (SEQ ID NO:196) 2B6^(b) V3DP47 3 VGVDRWYPADY (SEQ ID NO:197) 3F6^(c) V3 DP47 2 DLLDGSGAYFDY (SEQID NO:198) 2A2^(b) V3 DP46 0 DLDYGGNAGYFDL (SEQ ID NO:199) 2B10^(b) V3DP46 0 DLDYGGNAGYFDL (SEQ ID NO:200) 2E6^(b) V3 DP46 0 DYTANYYYYGMDV(SEQ ID NO:201) 3D1b V3 DP47 7 DLGYGSGTSSYYLDY (SEQ ID NO:202)Non-immune library Light Chain V_(L) CDR3 2A9^(b) Vκ1 L12A 6 QQANSFPRT(SEQ ID NO:203) 2B1^(b) Vκ1 L1 11 LQDYNGWT (SEQ ID NO:204) 2H6^(b) Vλ3DPL16 7 NSRDSSGNHVV (SEQ ID NO:205) 3C2^(b) Vλ3 DPL16 9 KSRDSRGNHLAL(SEQ ID NO:206) 2B6^(b) Vκ1 L12A 5 QQYHTISRT (SEQ ID NO:207) 3F6^(c) Vλ3DPL16 3 NSRDSSGNHVV (SEQ ID NO:208) 2A2^(b) Vλ3 DPL16 10 HSRDSSVTNLD(SEQ ID NO:209) 2B10^(b) Vλ3 DPL16 4 NSRDSSGNHQV (SEQ ID NO:210) 2E6^(b)Vλ3 DPL12 14 NSRDSSGVV (SEQ ID NO:211) 3D1^(b) Vλ3 DPL16 5 NSRDSSGNHVV(SEQ ID NO:212) Immune Library Heavy Chain Diff from Clone FamilySegment Genome V_(H) CDR3 3B8^(c) V_(H)1 V1-2 10 LATYYYFGLDV (SEQ IDNO:213) 3F10^(c) V_(H)1 V1-2 10 LATYYYFGLDV (SEQ ID NO:214) 2B11^(c)V_(H)1 DP10 11 GPWELVGYFDS (SEQ ID NO:215) 3A6^(c) V_(H)3 DP50 18EPDWLLWGDRGALDV (SEQ ID NO:216) 3D12^(c) V_(H)3 DP50 13 EPDWLLWGDRGALDV(SEQ ID NO:217) 2A1^(b) V_(H)3 DP50 14 EPDWLLWGDRGALDV (SEQ ID NO:218)Immune Library Light Chain Diff from Clone Family Segment Genome V_(L)CDR3 3B8^(c) Vκ1 DPK7 12 QQYNSYVYT (SEQ ID NO:219) 3F10^(c) Vκ1 DPK8 10QQLNSYPLT (SEQ ID NO:220) 2B11^(c) Vκ1 L12 11 QQLISYPLT (SEQ ID NO:221)3A6^(c) Vκ1 L12 8 QHYNTYPYT (SEQ ID NO:222) 3D12^(c) Vκ1 L12 10QHYNTYPYT (SEQ ID NO:223) 2A1^(b) Vκ1 L12 4 QHYNTYPYT (SEQ ID NO:224)^(a)Human germline VH, Vκ and Vλ segments have been assigned as detailedin the V-BASE database (MRC Centre for Protein Engineering, Cambridge,UK). Listed clones, with identical VH or VL CDR 3 regions, showeddifferent CDR 1, CDR 2 and framework regions, as indicated by theirdifferences from the germline genes; accession can be made throughGenBank with nos. AF090405-AF090420. ^(b)Library selected on BoNT/A.^(c)Library selected on BoNT/A HC.

Epitope Mapping of BoNT/A HC Specific Antibody Fragments

BoNT/A HC binding scFv were epitope mapped to determine the number ofnon-overlapping epitopes recognized. Epitope mapping was performed usingsurface plasmon resonance in a BIAcore™ studying pairs of scFv atconcentrations resulting in near saturation of the chip surface and atleast 100 RU of scFv bound. The amount of scFv bound was determined foreach member of a pair, and then the two scFv were mixed together to givea final concentration equal to the concentration used for measurementsof the individual scFv. Antibodies recognizing identical epitopes showedminimal increase in RU bound when injected together (FIG. 9A), whilescFv recognizing different epitopes showed an additive increase in RU(FIG. 9B). As depicted in Tables 2 and 3, scFv 3A6, 3D12 and 2A1,referred to as cluster I, share high homology of the VH and VL genesegments (DP 50 and L12, respectively) and recognize overlappingepitopes. They differ in sequence only by mutations in the heavy andlight chain genes introduced by somatic mutations. The scFv 3B8 and3F10, referred to as cluster II, form a second set of antibodies bindingto a different epitope compared to cluster I. Clone 2B11, representing apossible unique epitope, could not be analyzed due to poor expressionlevels. When scFv antibodies derived from the non-immune library wereanalyzed, we found that all bound to unique epitopes, referred to asclusters III-XI as depicted in Table 3. Members of the non-immunelibrary (clusters III-XI) showed no overlapping binding with members ofthe immune library (clusters I and II). The epitopes recognized by boththe immune and non-immune scFv do not overlap with the epitopes bound bytwo previously reported murine scFv, C25 and S25 (Amersdorfer et al.(1997) Infect. Immun., 65:3743-3752).

Kinetic Measurements and Neutralization Assay

The kon and koff were measured using surface plasmon resonance in aBIAcore and used to calculate the equilibrium dissociation constant. ThescFv selected from the immune library had K_(d)'s of 3.69×10⁻⁸ and7.8×10⁻⁹ M, values comparable to those reported for monoclonal IgGproduced from hybridomas (Foote and Milstein (1991) Nature, 352:530-532)(Table 12). Non-immune scFv had lower Kd's ranging from 4.6×10⁻⁷ to2.61×10⁻⁸ M. To determine the ability of scFv to neutralize toxininduced neuroparalysis, in vitro studies were performed on onerepresentative member from each epitope cluster using phrenicnerve-hemidiaphragm preparations. Values were reported in time to 50%twitch reduction for BoNT/A alone and in the presence of 2.0×10⁻⁸ MscFv. As shown in Table 12 and FIGS. 10A and 10B, a significantdifference in neutralization of the different anti-BoNT/A H_(C) scFvswere found, depending on which library was used. From the immunelibrary, 3D12 (cluster I) significantly prolonged the time toneuroparalysis 1.5-fold, whereas 3F10 (cluster II) exhibited no effecton toxin neutralization. Representatives of the non-immune library(clusters III-XI) showed no protective effect in the hemidiaphragmassay, even after combination of all members of clusters III-XI at afinal concentration of 1.8×10⁻⁷ M. When using a combination of 3D12(cluster I) with a previous isolated murine scFv, C25 (Amersdorfer etal. (1997) Infect. Immun., 65:3743-3752), time to paralysis increasedsignificantly to 3.2-fold, demonstrating a synergistic effect on toxinneutralization. We observed similar synergy with murine scFv S25 and3D12 (data not shown).

TABLE 12 Affinities, binding kinetics, and in vitro toxin neutralizationresults of scFv selected from phage antibody libraries. Clus- K_(d)k_(on) k_(off) Paralysis Clone ter (M)^(a) (×10⁵ (Ms)⁻¹) (×10⁻³ s⁻¹)Time^(b) Immune Library 3D12^(c) I 3.69 × 10⁻⁸ 0.13 0.50   85 ± 5.0^(d)3F10^(c) II 7.80 × 10⁻⁹ 0.80 0.62   55 ± 5.0^(e) Non-Immune Library2B10^(f) III 1.29 × 10⁻⁷ 5.57 71.6 62.3 ± 6.7^(e) 2E6^(f) IV 1.93 × 10⁻⁷1.19 23.0 60.9 ± 8.2^(e) 2H6^(f) V 3.86 × 10⁻⁸ 2.20 8.50 63.0 ± 5.0^(e)2B1^(f) VI 1.07 × 10⁻⁷ 0.83 8.88 58.4 ± 4.0^(e) 2A9^(f) VII 2.61 × 10⁻⁸0.25 0.66 71.0 ± 3.0^(e) 2B6^(f) VIII 7.15 × 10⁻⁸ 1.09 7.80 61.9 ±5.0^(e) 3D1^(f) IX 4.60 × 10⁻⁷ 1.31 60.3 58.3 ± 3.8^(e) 3F6^(c) X 6.60 ×10⁻⁸ 4.69 30.9 60.4 ± 3.6^(e) 3C2^(f) XI 3.90 × 10⁻⁸ 2.10 82.0 61.9 ±4.8^(e) Murine Library S25 XII 7.30 × 10⁻⁸ 0.11 0.82   85 ± 10^(d) C25XIII 1.10 × 10⁻⁹ 3.0 0.33  151 ± 12^(d) Combination C25 +  218 ± 22^(g)S25 C25 +  179 ± 2.3^(d) 3D12 Non-immune scFv   65 ± 2.3^(g) (ClustersIII-XI) BoNT/A pure toxin (control)   56 ± 3.8 ^(a)The variables kon andkoff were measured by surface plasmon resonance and Kd calculated askoff/kon. ^(b)Time (min) to 50% twitch reduction in mouse hemidiaphragmassay using 20 nM scFv + 20 pM BoNT/A, compared to time for BoNT/Aalone. Each value is the mean ± S.E.M. of at least three observations.^(c)Library selected on BoNT/A. ^(d)P < 0.01 compared to BoNT/A. ^(e)Notsignificant. ^(f)Library selected on BoNT/A HC. ^(g)P < 0.01 compared toBoNT/A HC.Discussion

We previously demonstrated that immunization of mice with therecombinant binding domain of BoNT/A HC directs the immune responsetowards generation of antibodies which bind epitope(s) involved in HCbinding to presynaptic toxin receptors (Amersdorfer et al. (1997)Infect. Immun., 65:3743-3752). These experiments indicated thatneutralization of toxin by scFv could be correlated to both scFvaffinity and ability to compete with the holotoxin for receptor bindingsites. Here we have carried out a more systematic approach by usingimmune and non-immune phage display libraries to map human humoralimmune and non-immune responses to BoNT/A. The source of antibody genesfor the two antibody libraries were (a) PBL of a human volunteerimmunized with pentavalent toxoid (A-E) and (b) non-immune peripheralblood lymphocytes and spleenocytes. One limitation of this approach isthe extent of which one immune human donor used for these studiesrepresents broad genetic diversity generated upon exposure to botulism.The fact that the humoral immune response in mice and human resulted ina rather limited number of protective epitopes, suggests significantconservation of antigenic epitopes conferring protection. The selectionprocedure involved panning both combinatorial libraries against fourimmobilized botulinum neurotoxins, serotypes A, B, C, and E. After threeto four panning cycles, antibodies against each serotype were obtainedfrom both libraries, with decreasing frequency in this order, BoNT/A,BoNT/B, BoNT/C and BoNT/E. Similar frequency of binders was alsoobserved for the non-immune library, with the exception of BoNT/B. Theseresults correlate with the findings of Siegel (Siegel (1989) J ClinMicrobiol., 26:2351-2356), where they studied serum specimens from 25human recipients of botulinum pentavalent toxoid. Immunogenicity of thevarious serotypes was determined by a mouse serum neutralizationbioassay—serotype A ranged between 5.7 and 51.6 IU/ml, followed byserotype B from 0.78 to 18 IU/ml and serotype E, from 0.61 to 10 IU/ml.

Human immunization with toxoid resulted in production of antibodiesdirected largely against the toxin light chain, with fewer antibodiesbinding HC. Similar results were observed after immunization of micewith BoNT/A HC followed by holotoxin boosts. Since antibodyneutralization activity results largely from blockade of cellularreceptor binding by HC, these analyses indicate that an HC vaccine willbe more protective than a toxin based vaccine, as more HC antibodies aregenerated. Human immune HC scFv recognized at least two non-overlappingepitopes. The scFv binding one of these epitopes (cluster I) couldneutralize toxin in vitro. Potency of toxin neutralization increasedwhen scFv binding cluster I were combined with immune mouse scFv bindingeither one of two non-overlapping HC epitopes. This result suggests thatHC docks with either multiple cellular receptors, or docking occurs overa broad surface area (Mullaney et al. (2001) Infect Immun.,69:6511-6514).

The repertoire of human scFv recognizing HC was extended to a range ofother epitopes (clusters III-XI) by selecting a large non-immune libraryon BoNT/A. Interestingly, this result is consistent with the conceptthat the primary immune repertoire contains antibodies capable ofrecognizing much of the solvent accessible area of an antigen, but thatimmunization directs this recognition to a limited number ofimmunodominant epitopes. All of the antibodies obtained from thenon-immune library, however, were directed against non-neutralizingepitopes (or at least did not neutralize toxin in vitro). Oneexplanation for the failure of neutralization could be due to lowaffinity of the antibodies for the HC domain (e.g. 2B10, 2E6, 2B1, 3D1),ranging from 107 to 460 nM compared to the high affinity interaction ofthe toxin to its receptor(s), which is 0.3-2.3 nM (Schengrund (1999) JToxicol Toxin Rev., 18:35-44).

In conclusion, we report here the successful isolation of specific humanantibodies toward botulinum neurotoxins and their subdomains usingcombinatorial libraries prepared from immune and non-immune humandonors. The use of phage display to screen the antibody repertoire ofany person with infectious diseases or pathogens allows us to access avery large pool of human monoclonal antibodies with therapeutic andresearch potential.

Example 4 Neutralizing Antibodies Evolved for Higher Affinity

To improve detection and treatment of botulism, molecular evolution andyeast display was used to increase the affinity of two neutralizingsingle chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A),HuC25 and 3D12.

Affinity Maturation of the mAb HuC25

The affinity of HuC25 for BoNT/A was sequentially increased using aseries of mutant yeast display libraries (FIG. 20). First, the HuC25gene was subcloned into the yeast display vector pYD2 as a NcoI-NotIfragment. The scFv gene successfully displayed on the yeast surface andthe KD of the displayed scFv for pure BoNT/A was determined by flowcytometry to be 8.44×10⁻¹⁰ M (FIG. 21). This is comparable to the K_(D)measured for purified HuC25 scFv binding to recombinant BoNT/A HC aspreviously measured using SPR in a BIAcore (1.4×10⁻⁹ M). The HuC25 scFvgene was then randomly mutated by PCR using error prone conditions andthe resulting gene repertoire cloned into pYD2 using gap repair tocreate a library of 2.0×10⁵ transformants (FIG. 20). The library wasgrown, induced, and then analyzed by flow cytometry for frequency ofscFv display (27%) and antigen binding (3.65%).

The library was then subjected to four rounds of selection usingdecreasing concentrations of pure BoNT/A. The scFv gene was PCRamplified from 6 individual colonies obtained after the final round ofsorting, revealing the presence of 1 unique sequence, AR1 (FIG. 18). TheAR1 clone was grown, scFv display induced, and the KD of the displayedscFv for BoNT/A was measured to be 1.69×10⁻¹⁰ M, a 5 fold improvementfrom HuC25 (FIG. 21).

To increase affinity further, two mutant yeast display libraries wereconstructed based on the sequence of AR1. For one library, the AR1 scFvgene was randomly mutated by using error prone PCR; for the secondlibrary, site directed mutagenesis was used to diversify four aminoacids (SNED) in the L3. This loop was selected for mutagenesis sincethis it was shown from the selection of AR1 that mutations here couldincrease affinity and it was likely that the error prone method had notfully sampled mutations in this loop. Four rounds of selection wereperformed for each library, with a final round of off rate selectionperformed by labeling with purified BoNT/A followed by a 12 hourdissociation in the presence of BoNT/A binding domain (HC) to preventrebinding. BoNT/A labeled yeast were then sorted using a mAb (7C1) whichbound the catalytic domain of the toxin. Screening of individualcolonies from the final round of sorting revealed only wild type AR1sequence for the site directed library, suggesting that L3 was alreadyoptimized. From the error prone library, a single unique clone wasisolated (AR2, FIG. 18), whose KD as a yeast displayed scFv wasdetermined to be 6.14×10¹¹ M, a 2.8 fold increase from the parental AR1(FIG. 21).

Additional yeast display libraries were created to further increase theaffinity of AR2. These included a library where random mutations wereintroduced into the AR2 gene and two site directed libraries based onthe sequence of AR2 which diversified either five amino acids in the H1or 4 amino acids in L2. Libraries were selected on pure BoNT/A using thestrategy described for the selection of AR1, individual coloniessequenced, and the affinities of the unique yeast displayed scFvmeasured. No clones of higher affinity were identified from the errorprone library or the library of L2 mutants. Two clones of higheraffinity were identified from the H1 library (FIG. 18), AR3 and AR4 (KDsof 1.9 and 2.3×10⁻¹¹ M, an approximate three fold increase in affinityfrom AR2, FIG. 21).

Affinity Maturation of the mAb 3D12

For affinity maturation, the 3D12 scFv gene was cloned as a NcoI-NotIfragment from the phagemid vector pCANTAB5E into the yeast displayvector pYD2. Random mutations were then introduced into the 3D12 scFvgene using PCR under error prone conditions and the resulting generepertoire cloned into pYD2 using gap repair to create a library of2.1×10⁶ transformants. The library was grown, induced, and then analyzedby flow cytometry for frequency of scFv display (27%) and antigenbinding (3.65%). The library was then subjected to five rounds ofselection using decreasing concentrations of BoNT/A. A final round ofoff rate selection was then performed by labeling with purified BoNT/Afollowed by a 15 hour dissociation in the presence of BoNT/A bindingdomain (HC) to prevent rebinding. BoNT/A labeled yeast were then sortedusing a mAb (7C1) which bound the catalytic domain of the toxin. ThescFv gene was PCR amplified from 6 individual colonies obtained afterthe final round of sorting, revealing the presence of 3 unique sequences(Table 13, clones 3-1 (also known as RAZ1, FIG. 19A), 3-8, and 3-10).Each unique clone was grown, scFv display induced, and the KD of thedisplayed scFv for BoNT/A measured using flow cytometry, along with thewild type 3D12 scFv. All three mutant scFv had higher affinity than thewild type 3D12 scFv. For the highest affinity scFv (RAZ1, KD in FIG.21), mutations were located entirely within the VL, in CDRs 1, 2, and 3(FIG. 19A).

TABLE 13 Amino acid sequences for affinity matured and/or modifiedantibodies. Clone Framework CDR1 Framework 2 CDR2 Heavy Chains HuC25QVQLQESGGGLVQPGGSLRLSC DYYMY (SEQ WVRQAPGKGLEW TISDGGSYTYYPD AASGFTFS(SEQ ID ID NO:226) VA(SEQ ID SVKG (SEQ ID NO:225) NO:227) NO:228) AR1QVQLQESGGGLVQPGGSLRLSC DYYMY(SEQ WVRQAPGKGLEW TISDGGSYTYYPD AASGFTFS(SEQ ID ID NO:230) VA (SEQ ID SVKG (SEQ ID NO:229) NO:231) NO:232) AR2QVQLQESGGGLVQPGGSLRLSC DHYMY (SEQ WVRQAPGKGLEW TISDGGSYTYYPD AASGFTFS(SEQ ID ID NO:234) VA (SEQ ID SVKG (SEQ ID NO:233) NO:235) NO:236)WR1(V) QVQLQESGGGLVQPGGSLRLSC DHYMY (SEQ WVRQAPGKGLEW TISDGGSYTYYPDAASGFTSS (SEQ ID ID NO:238) VA (SEQ ID SVKG (SEQ ID NO:237) NO:239)NO:240) WRI(T) QVQLQESGGGLVQPGGSLRLSC DHYMY (SEQ WVRQAPGKGLEWTISDGGSYTYYPD AASGFTSS (SEQ ID ID NO:242) VA (SEQ ID SVKG (SEQ IDNO:241) NO:243) NO:244) 3D12 QVQLVQSGGGVVHPGRSLKLSC DYDMH (SEQWVRQAPGKGLEW VMWFDGTEKYSAE AGSGFTFS (SEQ ID ID NO:246) VA (SEQ IDSVKG(SEQ ID NO:245) NO:247) NO:248) RAZ1 QVQLVQSGGGVVHPGRSLKLSC DYDMH(SEQ WVRQAPGKGLEW VMWFDGTEKYSAE AGSGFTFS (SEQ ID ID NO:250) VA (SEQ IDSVKG (SEQ ID NO:249) NO:251) NO:252) 3-8 QVQLVQSGGGVVHPGRSLKLSC DYDMH(SEQ WVRQAPGKGLEW VIWFDGTEKYSAE AGSGFTFS (SEQ ID ID NO:254) VA (SEQ IDSVKG (SEQ ID NO:253) NO:255) NO:256) 3-10 QVQLVQSGGGVVHPGRSLKLSC DYDMH(SEQ WVRQAPGKGFEW VMWFDGTEKYSAE AGSGFTFS (SEQ ID ID NO:258) VA (SEQ IDSVKG (SEQ ID NO:257) NO:259) NO:260) ING1 QVQLQQSGGGLVQPGGSLRLSC NYAMT(SEQ WVRQAPGKGLEW SISVGGSDTYYAD AASGFTFS (SEQ ID NO:40) ID NO:41) VS(SEQ ID SVKG (SEQ ID NO:42) NO:43) Framework 3 CDR3 Framework 4 HuC25RFTISRDNSKNTLYLQMNSLRA YRYDDAMDY WGQGTLVTVSS EDTAMYYCSR (SEQ ID (SEQ ID(SEQ ID NO:261) NO:262) NO:263) AR1 RFTISRDNSKNTLYLQMNSLRA YRYDDAMDYWGQGTLVTVSS EDTAIYYCSR(SEQ ID (SEQ ID (SEQ ID NO:264) NO:265) NO:266)AR2 RFTTSRDNSKNTLYLQMNSLRA YRYDDAMDY WGQGTLVTVSS EDTAIYYCSR (SEQ ID (SEQID (SEQ ID NO:267) NO:268) NO:269) WR1(V) RFTVSRDNSKNTLYLQMNSLRAYRYDDAMDY WGQGTLVTVSS EDTAIYYCSR (SEQ ID (SEQ ID (SEQ ID NO:270) NO:271)NO:272) WR1(T) RFTTSRDNSKNTLYLQMNSLRA YRYDDAMDY WGQGTLVTVSS EDTAIYYCSR(SEQ ID (SEQ ID (SEQ ID NO:273) NO:274) NO:275) 3D12RFTISRDNSKNTLFLQMNSLRA EPDWLLWGDRG WGQGTTVTVSS DDTAVYYCAR (SEQ ID ALDV(SEQ ID (SEQ ID NO:276) NO:277) NO:278) RAZ1 RFTISRDNSKNTLFLQMNSLRAEPDWLLWGDRG WGQGTTVTVSS DDTAVYYCAR (SEQ ID ALDV (SEQ ID (SEQ ID NO:279)NO:280) NO:281) 3-8 RFTISRDNSKNTLFLQMNSLRA EPDWLLWGDRG WGQGTTVTVSSDDTAVYYCAR (SEQ ID ALDV (SEQ ID (SEQ ID NO:282) NO:283) NO:284) 3-10RFTISRDNSKNTLFLQMNSLRA EPDRLLWGDRG WGQGTTVTVSS DDTAVYYCAR (SEQ ID ALDV(SEQ ID (SEQ ID NO:285) NO:286) NO:287) ING1 RFTVSRDNSKNTLLLQMNSLRAVRTKYCSSLSC WGQGTRVTVSS EDTAVYYCAK (SEQ ID FAGFDS (SEQ (SEQ ID NO:68) IDNO:69) NO:70) Clone Framework 1 CDR1 Framework 2 CDR2 Light Chains HuC25EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQ C (SEQ IDNO:288) SFMQ (SEQ ID LIY (SEQ ID ID NO:291) NO:289) NO:290) AR1EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQ C (SEQ IDNO:292) SFMQ (SEQ ID LIY (SEQ ID ID NO:295) NO:293) NO:294) AR2EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQ C (SEQ IDNO:296) SFMQ (SEQ ID LIY (SEQ ID ID NO:299) NO:297) NO:298) WR1(V)EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQ C (SEQ IDNO:300) SFMQ (SEQ ID LIY (SEQ ID ID NO:303) NO:301) NO:302) WR1(T)EIVLTQSPATLSLSPGERATIS RASESVDSYGH WYQQKPGQAPRL RASNLEP (SEQ C (SEQ IDNO:304) SFMQ (SEQ ID LIY (SEQ ID ID NO:307) NO:305) NO:306) 3D12DIVMTQSPSTLSASVGDRVTIT RASQSISSWLA WYQQKPGKAPKL EASSLES (SEQ C (SEQ IDNO:308) (SEQ ID LMY (SEQ ID ID NO:311) NO:309) NO:310) RAZIDIVMTQSPSTLSASVGDRVTIT WASQSISSRLA WYQQKPGKAPKL EATSLGS (SEQ C (SEQ IDNO:312) (SEQ ID LMY (SEQ ID ID NO:315) NO:313) NO:314) 3-8DIVMTQSPSTLSASVGDRVTIT RASQSISSWLA WYQQKPGKAPKL GASSLGS (SEQ C (SEQ IDNO:316) (SEQ ID LMY (SEQ ID ID NO:319) NO:317) NO:318) 3-10DIVMTQSPSTLSASVGDRVTIT RASQSISSWLA WYQQKPGKAPKL EASSLGR (SEQ C (SEQ IDNO:320) (SEQ ID LMY (SEQ ID ID NO:323) NO:321) NO:322) ING1DIVMTQSPSSLSASVGDRVTIT RASQSISSYLN WYQQKPGKAPKL AASSLQS (SEQ C (SEQ IDNO:96) (SEQ ID LIY (SEQ ID ID NO:99) NO:97) NO:98) Clone Framework 3CDR3 Framework 4 HuC25 GIPARFSGSGSGTDFTLTISSL QQSNEDPFT FGQGTKVEIKREPEDFAVYYC (SEQ ID (SEQ ID (SEQ ID NO:324) NO:325) NO:326) AR1GIPARFSGSGSGTDFTLTISSL QQGNEVPFT FGQGTKVEIKR EPEDFAVYYC (SEQ ID (SEQ ID(SEQ ID NO:327) NO:328) NO:329) AR2 GIPARFSGSGSGTDFTLTISSL QQGNEVPFTFGQGTKVEIKR EPEDFAVYYC (SEQ ID (SEQ ID (SEQ ID NO:330) NO:331) NO:332)WR1(V) GIPARFSGSGSGTDFTLTISSL QQGNEVPFT FGQGTKVEIKR EPEDFAVYYC (SEQ ID(SEQ ID (SEQ ID NO:333) NO:334) NO:335) WR1(T) GIPARFSGSGSGTDFTLTISSLQQGNEVPFT FGQGTKVEIKR EPEDFAVYYC (SEQ ID (SEQ ID (SEQ ID NO:336) NO:337)NO:338) 3D12 GVPSRFSGSGSGTEFTLTISSL QHYNTYPYT FGQGTKLEIKR QPDDFAAYYC(SEQ ID (SEQ ID (SEQ ID NO:339) NO:340) NO:341) RAZ1GVPSRFSGSGSGTEFTLTISSL QHYDTYPYT FGQGTKLEIKR QPDDFAAYYC (SEQ ID (SEQ ID(SEQ ID NO:342) NO:343) NO:344) 3-8 GVPSRFSGSGSGTEFTLTISSL QHYNTYPYTFGQGTKLEIKR HPDDFAAYYC (SEQ ID (SEQ ID (SEQ ID NO:345) NO:346) NO:347)3-10 GVPSRFSGSGSGTEFTLTISSL QHYSTYPYT FGQGTKLEIKR QPDDFAAYYC (SEQ ID(SEQ ID (SEQ ID NO:348) NO:349) NO:350) ING1 GVPSRFSGSGSGTDFTLTISSLQQSYSTPRTT FGGGTKVDIKR QPEDFATYYC (SEQ ID (SEQ ID (SEQ ID NO:124)NO:125) NO:126) *Sequence for complete heavy chain is heavy chainframework 1 + CDR1 + framework 2 + CDR2 + framework 3 + CDR3 + framework4. Sequence for complete light chain is light chain framework 1 + CDR1+ framework 2 + CDR2 + framework 3 + CDR3 + framework 4.Impact of Conversion of Yeast Displayed scFv to IgG on Affinity

For many immunologic assays, as well as in vivo neutralization studies,it is necessary to utilize IgG. We therefore converted HuC25, AR1, AR2,AR3, AR4, 3D12, and RAZ1 to full length IgG consisting of the humangamma 1 constant region and the human kappa constant region bysequential cloning of the VH and Vk genes into a mammalian expressionvector driven by dual CMV promoters. Stable CHO DG44 cell lines wereestablished for each of the 7 antibodies and IgG was purified from cellculture supernatant in yields of 5-20 mg/L for six of the sevenantibodies. We were unable to express any significant quantities of theAR3 IgG.

The affinities of each IgG was measured kinetic exclusion analysis(Kinexa). The affinities of the HuC25 family of mutants and of RAZ1 weresignificantly higher as IgG than yeast displayed scFv, but the relativeincrease in affinity of the IgG, were consistent with the relativeaffinities determined on yeast displayed scFv (Table 14). For examplethe AR4 scFv had a 37 fold higher affinity than HuC25 scFv by yeastdisplay and the AR4 IgG had a 34 fold higher affinity than the HuC25 IgGas measured by Kinexa. The RAZ1 scFv had a 45 fold higher affinity than3D12 scFv by yeast display and the RAZ1 IgG had a 35 fold higheraffinity than the 3D12 IgG as measured by Kinexa.

TABLE 14 Affinity of antibodies on A1 and A2 toxins. Antibody K_(D) onHall (A1) toxin K_(D) on Honey (A2) toxin HuC25 1.24 nM   250 nM  AR1200 pM  100 nM  AR2 47 pM ND WR1 (V) 450 pM  9.0 nM  WR1 (T) 310 pM  3.7nM  3D12 940 pM  2.2 nM  3D12.3-1 (RAZ1) 17 pM 70 pM 3D12.3-8 21 pM 67pM 3D12.3-10 28 pM 81 pMImpact of Affinity on Detection of BoNT/A by Flow Cytometry

Higher affinity scFv displayed on yeast were able to detectsignificantly lower concentrations of BoNT/A compared to lower affinityyeast displayed scFv (FIG. 22). The highest affinity scFv (AR4) was ableto detect as little as 0.1 pM of BoNT/A, a value lower than thatreported for other non-amplified BoNT detection systems. Thus theresults validate the utility of increasing antibody affinity to increasedetection sensitivity.

Impact of Affinity on Neutralization of BoNT/A

The wild type and higher affinity antibodies were studied in the in vivomouse neutralization assay. For a single antibody, higher affinity ledto small (approximately 2 fold) increase in protection of micechallenged with intraperitoneal BoNT/A (FIG. 23), with the highestaffinity AR4 antibody providing complete protection against 100 mouseLD50s of toxin but not against 200 LD50s. When two antibodies werecombined, protection increased significantly, with the combination ofAR4+3D12 providing approximately a 2 fold increase in protection, from2500 LD50s to 5000 LD50s. When RAZ1 was substituted for 3D12 in theantibody pairs, protection was seen out to 10,000 mouse LD50s for thecombination of AR4 and RAZ1. Thus the data indicate that using higheraffinity antibodies in antibody combinations leads to more potent toxinneutralization. This is even more clear for combinations of threeantibodies (Table 15).

TABLE 15 Potency of neutralization of antibody combinations. 1000 25005000 10,000 20,000 40,000 LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ LD₅₀ HuC25:B4:3D12,10/10 20/20 50 μg HuC25:B4:3D12, 10/10 10/10 1/10 0/10 10 μgHuC25:B4:RAZ1,  8/10 10 μg CR1:RAZ1:ING1, 10/10  5 μg CR1:RAZ1:ING1,18/20  2 μg CR1:RAZ1:ING1, 8/10 1 μg CR1:RAZ1:ING1, 3/10 0.5 μg

Here the replacement of 3D12 with the higher affinity RAZ1 in acombination of HuC25/B4/3D12 or RAZ1, provides complete protection at a10,000 LD50 challenge dose of toxin. With the wildtype 3D12 in thecombination, no mice survive challenge at 10,000 LD50s. Replacing B4with the higher affinity ING1 and HuC25 with the higher affinity CR1allows a decrease in the antibody dose from 50 ug to 1 ug with still 80%survival at a 10,000 LD50 challenge dose of toxin. Thus increasing theaffinity of single antibodies used in antibody combinations increasespotency and allows for a decrease in antibody dose.

Example 5 Sequence Variation Within Botulinum Neurotoxin SerotypesImpacts Antibody Binding And Neutralization

Materials and Methods

Toxin Gene Sequences:

The NCBI databases and Medline were searched to identify published orarchived sequences of botulinum neurotoxin genes or proteins. Theneurotoxin gene of Clostridial strain FRI-A2H was sequenced for thiswork (manuscript in preparation). The neurotoxin gene sequence ofClostridial strain was a gift of Michael Peck. Gene sequences wereentered into Vector NTI (Invitrogen, San Diego, Calif.), translated,classified by serotype and aligned. Phylogenetic trees were constructedusing ClustalW.

Toxins and Antibodies:

Purified pure and complexed botulinum neurotoxins A1 (Hall hyper) and A2(FRI-A2H) were purchased from Metabiologics Inc (Madison, Wis.).Antibodies S25 and C25 were derived from a single chain Fv phage displaylibrary constructed from the V-genes of an immunized mouse (Amersdorferet al. (1997) Infect. Immun. 65: 3743-3752; Nowakowski et al. (2002)Proc. Natl. Acad. Sci. USA, 99: 11346-50). Antibody 3D12 was derivedfrom a single chain Fv phage display library constructed from theV-genes of an immunized human volunteer donor (Amersdorfer et al. (2002)Vaccine 20: 1640-1648; Amersdorfer et al. (1997) Infect. Immun. 65:3743-3752). Antibody B4 was derived from a single chain Fv phage displaylibrary constructed from the V-genes of an immunized mouse transgenicfor the human immunoglobulin locus (Xenomouse), (I. Geren and J. D.Marks, submitted). The V-genes of each of these four antibodies werecloned into a mammalian expression vector containing human IgG1 andkappa constant regions as previously described (Nowakowski et al. (2002)Proc. Natl. Acad. Sci. USA, 99: 11346-50). Stable CHO DG44 cell lineswere established and IgG purified using protein G as previouslydescribed (Nowakowski et al. (2002) Proc. Natl. Acad Sci. USA, 99:11346-50). Antibody purity and concentration was determined by SDS-PAGEand absorbance at 280 nm. Antibodies 9D8 (murine IgG1/kappa) and 7C1(murine IgG1/kappa) were derived from hybridomas generated from miceimmunized with rBoNT/A H_(C) and boosted with BoNT/A toxin. IgG werepurified from hybridoma supernatants using protein G and purity andconcentration determined by SDS-PAGE and BCA assay (Pierce ChemicalCo.). For subsequent studies, IgG antibodies were stored in PBS, pH 7.4at approximately 1-3 mg/ml.

Toxin Capture ELISA:

For toxin capture ELISA, 96 well microtiter plates (Immunolon 2,Dynatech) were coated with antibody at 2 μg/ml overnight at 4° C. Afterblocking for 30 minutes in 5% skim milk-PBS, toxins were applied inhalf-log dilutions from 100 nM to 1 pM in duplicate and A incubated for90 minutes at 37° C. Plates were washed and incubated with equineanti-BoNT antibody (PerImmune), diluted to 0.2 IU/mi, for 60 minutes,followed by washing and incubation with a 1:1000 dilution of goatanti-horse antibody conjugated to horseradish peroxidase (KPL) for 60minutes. Plates were developed with ABTS (KPL). Average absorbance at405 nm after subtraction of background control was plotted against toxinconcentration.

Measurement of Antibody Affinity for Toxin:

IgG association and dissociation rate constants for purified BoNT/A1 orA2 toxins were measured using surface plasmon resonance in a BIAcore1000 (Pharmacia Biosensor) and used to calculate the KD as previouslydescribed (Nowakowski et al. (2002) Proc. Natl. Acad. Sci. USA, 99:11346-11350). Briefly, approximately 100-400 RU of purified IgG (10-20ug/ml in 10 mM acetate, pH 3.5-4.5) was coupled to a CM5 sensor chipusing NHS-EDC chemistry. The association rate constant for purifiedBoNT/A1 or A2 neurotoxins was measured under continuous flow of 15ul/min using a concentration range of 50 nM to 800 nM toxin. Associationrate constant (kon) was determined from a plot of (ln(dR/dt))/t vs.concentration. The dissociation rate constant (koff) was determined fromthe dissociation part of the sensorgram at the highest concentration oftoxin analyzed using a flow rate of 30 μl/min to prevent rebinding. KDwas calculated as koff/kon.

Measurement of In Vivo Toxin Neutralization:

Fifty μg of the appropriate IgG were added to the indicated number ofmouse LD₅₀s of BoNT/A1 neurotoxin complex (Hall strain) or BoNT/A2neurotoxin complex (FRI-A2H strain) in a total volume of 0.5 ml ofgelatin phosphate buffer and incubated at RT for 30 min. For pairs ofmAbs, 25 μg of each mAb was added, and for the combination of 3 mAbs,16.7 μg of each mAb was added. The mixture was then injectedintraperitoneally into female CD-1 mice (16-22 grams on receipt). Micewere studied in groups of 10 and were observed at least daily. The finaldeath tally was determined 5 days after injection. Studies using micewere conducted in compliance with the Animal Welfare Act and otherFederal statutes and regulations relating to animals and experimentsinvolving animals and adhere to principles stated in the Guide for theCare and Use of Laboratory Animals, National Research Council, 1996. Thefacility where this research was conducted is fully accredited by theAssociation for Assessment and Accreditation of Laboratory Animal CareInternational.

Results

Sequence Variation Within Botulinum Neurotoxin Serotypes

To determine the extent of sequence variability within toxin serotypes,the literature was searched revealing 60 published neurotoxin sequences.This data included 49 complete toxin gene sequences and 11 partial toxingene sequences (Table 16). The 49 complete sequences were classified byserotype, aligned, and the extent of sequence identity determined (Table17 and FIG. 11). Of the 49 sequences analyzed, there were 7 BoNT/A, 9BoNT/B, 6 BoNT/C, 5 BoNT/D, 17 BoNT/E, 4 BoNT/F, and 1 BoNT/G. Withinserotypes, two types of toxin gene sequences were observed; those thatwere virtually identical to each other (vide infra) and those thatdiffered by at least 2.6% at the amino acid level. Such sequencevariability was observed within all six serotypes where more than 1toxin gene had been sequenced (BoNTs A, B, C, D, E, and F). Withinserotypes, variability ranged from a high of 32% for BoNT/F to a low of2.6% for BoNT/E (Table 17). Three BoNT C/D and two BoNT D/C mosaicstrains were sequenced. These strains typically contained light chainsand N terminal heavy chains that matched their parental serotype, withthe terminal third of the neurotoxin sequence having strong, but notabsolute, identity with the alternative serotype of the mosaics (Table16).

TABLE 16 Clostridial strains used in sequence analyses. Accessionnumbers are from the NCI nucleotide database. serotype subtype strain(s)accession # reference(s) A A1 NCTC 2916 X52066 Thompson, 1990 [1] 62AM30196 Binz, 1990b [2] ATCC 3502 (Dr. Michael Peck, unpublished) Hallhyper AF461540 Dineen, 2003 [3] Hall Allergan AF488749 Zhang, 2003 [4]A2 Kyoto-F X73423 Willems, 1993 [5] FRI-A2H (Bradshaw et al,unpublished) B B1 Danish M81186 Whelan, 1992 [6] BGB Kirma, 2004 [7]okra Ihara, 2003 [8] B2 strain 111 AB084152 Ihara, 2003 [8]nonproteolytic B Eklund 17B X71343 Hutson, 1994 [9] bivalent B CDC 588AF300465 Kirma, 2004 [7] CDC 593 AF300466 Kirma, 2004 [7] CDC 1436AF295926 Kirma, 2004 [7] CDC 3281 Y13630 Santos-Buelga, 1998 C C1Stockholm X66433 Hauser, 1990; X62389 Kimura, 1990 [10, 11] C 468 X72793Hauser, 1994 [12] Yoichi AB061780 Sagane, 2001 [13] C/D 6813 D49440Moriishi, 1996 [14] 6814 AB037166 TW/2003 AY251553 D D BVD/-3 X54254Binz, 1990 [15] CB-16 S49407 Sunagawa, 1992 [16] 1873 AB012112 Nakajima,1998 [17] D/C South Africa D38442 Moriishi, 1996 [14] D 4947 AB037920Kouguchi, 2002 [18] E E botulinum NCTC 11219 X62683 Whelan, 1992 [19]Beluga X62089 Poulet, 1992 [20] 35396 AB082519 Tsukamota, 2002 [21] Ebutyricum BL5262, BL6340 X62088 Poulet, 1992 [20] X62088 BL5520 Q9FAR6Wang, 2000 [22] KZ 1886 AB037708 Wang, 2000 [22] KZ 1887 AB037709 Wang,2000 [22] KZ 1889 AB037710 Wang, 2000 [22] KZ 1890 AB037711 Wang, 2000[22] KZ 1891 AB037712 Wang, 2000 [22] KZ 1897 AB037706 Wang, 2000 [22]KZ 1898 AB037707 Wang, 2000 [22] KZ 1899 AB037705 Wang, 2000 [22] LCL063 AB037713 Wang, 2000 [22] LCL 095 AB037714 Wang, 2000 [22] LCL 155AB037704 Wang, 2000 [22] F proteolytic F Langeland X81714 Hutson, 1994[9]; L35496 Elmore, unpublished nonproteolytic F Eklund 202F M92906East, 1992 [23] F baratii ATCC 43756 X68262 Thompson, 1993 [24] bivalentF CDC 3281 (Bf) Y13631 Santos-Buelga, 1998 [25] G G 113/30 X74162Campbell, 1993 [26] [1] Thompson et al. (1990) Eur. J. Biochem., 189(1):73-81. [2] Binz et al. (1990) J. Biol. Chem., 265(16): 9153-9158. [3]Dineen et al. (2003) Cur.r Microbiol., 46(5): 345-352. [4] Zhang et al.(2003) Gene, 315: 21-32. [5] Willems et al.(1993) Res. Microbiol.,144(7): 547-556. [6] Whelan et al. (1992) Appl. Environ. Microbiol.,58(8): 2345-2354. [7] Kirma et al. (2004) FEMS Microbiol. Lett., 231(2):159-164. [8] Ihara et al. (2003) Biochim. Biophys. Acta, 1625(1): 19-26.[9] Hutson et al. (1994) Curr. Microbiol., 28(2): 101-110. [10] Hauseret al. (1990) Nucleic Acids Res., 18(16): 4924. [11] Kimura et al.(1990) Biochem. Biophys. Res. Commun., 171(3): 1304-1311. [12] Hauser etal. (1994) Mol. Gen. Genet., 243(6): 631-640. [13] Sagane et al. (2001)Biochem. Biophys. Res. Commun., 288(3): 650-657. [14] Moriishi et al.(1996) Biochim. Biophys. Acta, 1307: 123-126. [15] Binz et al. (1990)Nucleic Acids Res., 18(18): 5556. [16] Sunagawa et al. (1992) J. Vet.Med. Sci., 54(5): 905-913. [17] Nakajima et al. (1998) Microbiol.Immunol., 42(9): 599-605. [18] Kouguchi et al. (2002) J. Biol. Chem.,277(4): 2650-2656. [19] Whelan et al. (1992) Eur. J. Biochem., 204(2):657-667. [20] Poulet et al. (1992) Biochem. Biophys. Res. Commun.,183(1): 107-113. [21] Tsukamoto et al. (2002) Microb. Pathog., 33(4):177-184. [22] Wang et al. (2000) Appl. Environ. Microbiol., 66(11):4992-4997. [23] East et al. (1992) FEMS Microbiol. Lett., 75(2-3):225-230. [24] Thompson et al. (1993) FEMS Microbiol. Lett., 108(2):175-182. [25] Santos-Buelga et al. (1998) Curr Microbiol., 37(5):312-318. [26] Campbell et al. (1993) Biochim Biophys Acta, 1216(3):487-491.

The two toxin serotypes causing more than 90% of human botulism (BoNT/Aand B, (Control (1998) Botulism in the United States, 1899-1998.Handbook for epidemiologists, clinicians, and laboratory workers.Atlanta, Ga. U.S. Department of Health and Human Services, Public HealthService: downloadable at bt.cdc.gov/agent/botulism/index.asp wereanalyzed in more detail. Of the seven published BoNT/A toxin sequences,five (62A, NCTC 2916, ATCC 3502, and Hall hyper (Hall Allergan)) werevirtually identical (99.9-100% identity) and have been classified assubtype A1 (FIG. 12A). The other two BoNT/A sequences (Kyoto-F andFRI-A2H) were 100% identical and have been classified as subtype A2(FIG. 12A). The A1 toxins differed from the A2 toxins by 10.1%, with thegreatest difference in sequence in the receptor binding domain(C-terminal heavy chain, HC). (Table 18). Besides being greater innumber, the HC amino acid differences tended to be located in solventaccessible amino acids exposed on the toxin surface (FIG. 12B). A numberof these differences clustered around the putative ganglioside bindingsite (FIG. 12B). The sequence of the catalytic domain (light chain) wasmore conserved (Table 18), 1 with the differences more likely to beburied (FIG. 12B).

TABLE 17 Classification of Clostridial botulinum neurotoxin genesequences. Subtypes were defined as differing by at least 2.6% at theamino acid level. Minimum and maximum complete partial amino aciddifferences serotype sequences sequences subtypes within serotype A 7 210.1% B 9 3 4 3.6-7.7% C 6 2 24.0-24.2% D 5 2 23.7-23.9% E 17 6 32.6-5.1% F 4 2 4 10.7-31.6% G 1 1 total: 49 11 18

TABLE 18 Percent amino acid identity between BoNT A1 and A2 strains.holotoxin light chain heavy chain H_(N) H_(C) BoNT A1 versus 89.9 95.187.1 87.1 87.2 BoNT A2

The nine published BoNT/B sequences could be grouped into 4 subtypesbased on DNA and protein homology (FIG. 13). These groups included thebivalent BoNT/B (BoNT Ab 1436, BoNT Ab 588, BoNT Ab 593, and BoNT Bf3281), BoNT/B1 (BoNT/B Danish), BoNT/B2 (BoNT/B strain 111), and thenonproteolytic BoNT/B (BoNT/B Eklund). These toxins differed from eachother by 3.6% to 7.7% at the amino acid level, with greater differencesin the heavy chain compared to the light chain (Table 19).

TABLE 19 Percent amino acid identity among BoNT B strains. heavyholotoxin light chain chain H_(N) H_(C) BoNT B1 vs: BoNT B2 95.7 99.593.6 95.8 91.8 BoNT B np 92.8 97.7 90.2 92.5 88.2 BoNT B 96.0-96.498.9-99.1 94.6-94.9 94.3-95.0 94.7-94.9 bivalent

Impact of BoNT/A Toxin Sequence Variation and Antibody Binding

To determine the impact of BoNT/A toxin sequence variability on immunerecognition we measured the ability of six monoclonal antibodies raisedagainst BoNT/A1 to bind to BoNT/A1 and BoNT/A2 by capture ELISA. Bindingto both pure neurotoxin and neurotoxin complex was determined. Four mAbs(3D12, C25, B4, and S25) bound to non-overlapping epitopes on the BoNT/AHC, as determined by ELISA on recombinant HC. 3D12 and S25 have beenpreviously epitope mapped to the C-terminal subdomain of BoNT/A HC,while C25 recognizes a complex epitope formed by the two HC subdomains(Mullaney et al. (2001) Inf. Immun., 69: 6511-6514). One mAb (9D8) boundthe BoNT/A translocation domain (HN) as determined by ELISA onrecombinant HN (data not shown). One mAb (7C1) bound the BoNT/A lightchain, as determined by ELISA on recombinant light chain.

Three of the four antibodies which bound the BoNT/A HC showed a markedreduction in binding to BoNT/A1 toxin compared to BoNT/A2 toxin (FIG.14). In contrast, non HC binding mAbs showed comparable ELISA signals onboth A1 and A2 toxins (FIG. 15). To quantitate the difference in bindingto A1 and A2 toxins, the equilibrium dissociation constant and bindingrate kinetics were measured for the binding of each mAb to purified A1and A2 toxins (Table 17). All mAbs bound A1 toxin with high affinity (KDranging between 6 and 0.17 nM). The three mAbs which demonstrateddecreased binding to A2 toxin by capture ELISA (C25, S25, and B4) showeda 553 to more than 1200 fold reduction in affinity for A2 toxin comparedto A1 toxin. It was not possible to measure a KD for the B4 mAb bindingto BoNT/A2 due to very low affinity binding. The majority of thereduction in affinity was due to a large decrease in the associationrate constant (Table 20). In contrast, three mAbs (3D12, 9D8 and 7C1)showed comparable high affinity for both A1 and A2 toxins.

TABLE 20 Association (k_(on)) and dissociation (k_(off)) rate constantsand equilibrium dissociation constants (K_(d)) for BoNT/A IgG binding toBoNT/A1 and BoNT/A2. Association and dissociation rate constants weredetermined by surface plasmon resonance in a BIAcore and K_(D)calculated as k_(off)/k_(on). NM = not meas*urable BoNT/A1 BoNT/A2Antibody K_(d) (M⁻¹) k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹) K_(d) (M⁻¹) k_(on)(M⁻¹ s⁻¹) k_(off) (s⁻¹) C25 2.98 × 10⁻¹⁰  1.5 × 10⁶ 4.47 × 10⁻⁴ 1.65 ×10⁻⁷ 2.09 × 10⁴ 3.63 × 10⁻³ S25 1.69 × 10⁻⁹ 4.82 × 10⁵ 8.15 × 10⁻⁴ 2.14× 10⁻⁶ 1.34 × 10³ 2.87 × 10⁻³ 3D12 1.68 × 10⁻¹⁰ 1.45 × 10⁶ 2.44 × 10⁻⁴1.04 × 10⁻⁹ 3.48 × 10⁵ 3.62 × 10⁻⁴ B4  1.8 × 10⁻⁹  7.2 × 10⁵ 1.31 × 10⁻³NM NM NM 7C1  5.9 × 10⁻⁹ 2.89 × 10⁵ 1.71 × 10⁻³  5.1 × 10⁻⁹ 3.38 × 10⁵1.73 × 10⁻³ 9D8 1.21 × 10⁻⁹ 1.73 × 10⁵ 2.11 × 10⁻⁴  1.3 × 10⁻⁹ 2.08 ×10⁵ 2.73 × 10⁻⁴

-   -   Impact of Antibody Binding on Neutralization of A1 and A2        Neurotoxins

We previously studied the in vivo neutralization capacity of three mAbsdescribed here, 3D12, S25, and C25, for BoNT/A1 toxin. Despite showingsignificant in vitro neutralization of BoNT/A1, none of these three mAbsshowed significant in vivo protection of mice receiving 50 ug ofantibody and challenged with 20 mouse LD50s of BoNT/A1 (only 10-20%survival, (Nowakowski et al. (2002) Proc. Natl. Acad Sci. USA, 99:11346-11350)). Similarly, none of the remaining three mAbs reported hereshowed significant in vivo protection when mice were challenged with 20mouse LD₅₀s of BoNT/A1 (only 10-20% survival, data not shown). Since wepreviously reported significant synergy in in vivo protection when mAbswere combined, we studied the ability of mAb pairs and triplets toneutralize toxin in vivo. As previously observed, antibody pairs showedsignificantly greater BoNT/A1 neutralization than single mAbs, with evengreater potency observed for combinations of three mAbs (FIGS. 16 and17A). Synergy was observed for mAb pairs that included only 1 bindingdomain antibody (C25+9D8) or no binding domain antibodies (9D8+7C1)(FIG. 16) or combinations of three mAbs that included only one bindingdomain antibody (C25+9D8+7C1) (FIG. 17A). With respect to neutralizationof BoNT/A2 toxin, only mAb pairs or triplets containing mAbs which boundBoNT/A2 with high affinity showed significant synergy for neutralization(FIG. 17B). The most potent mAb triplet (3D12+9D8+7C1) was able tocompletely protect mice from a challenge of 10,000 mouse LD₅₀s of A1 orA2 toxin. While this combination (3D12+9D8+7C1) was not as potent forneutralization of A1 toxin as a combination of three binding domain mAbs(C25+3D12+B4), only one binding domain mAb bound A2 toxin with anyaffinity, and as a result the C25+3D12+B4 triplet neutralized less than200 mouse LD₅₀s of A2 toxin.

Discussion

Analysis of 49 complete published botulinum neurotoxin sequencesrevealed that within serotypes, toxin gene sequences were eithervirtually identical or differed from each other by at least 3.6% at theamino acid level. We have termed those toxins with this minimumdifference (3.6%) to be subtypes of a given serotype. Such analysisrevealed an average of 2.8 subtypes for the six serotypes where morethan one toxin gene has been sequenced (range 2-4 subtypes/serotype).While this analysis probably reveals the most frequent toxin subtypes,it is likely that additional toxin subtypes remain to be identified,given the relatively small number of toxin genes sequenced (on average 8toxin genes/serotype).

The importance of toxin subtypes is their impact on diagnostic tests andthe development of toxin therapeutics. Clearly, this level of nucleotidepolymorphism can affect DNA probe based assays such as PCR. Importantly,the extent of amino acid substitution can affect the binding ofmonoclonal antibodies used for ELISA and other immunologic baseddiagnostic tests. We have clearly shown that the 10% amino aciddifference between BoNT/A1 and BoNT/A2 subtypes has a dramatic effect onthe binding affinity and ELISA signals of three of six monoclonalantibodies analyzed. Interestingly, the kinetic basis for the reducedmAb affinity is largely due to a decrease in the association rateconstant, rather than an increase in the dissociation rate constant. Theimpact of the difference in toxin amino acid sequence on the binding ofpolyclonal antibody is unknown. Clearly, toxin assays based onimmunologic recognition will need to be validated using the differenttoxin subtypes.

The differences in binding affinity translate into significantdifferences in the potency of in vivo toxin neutralization. Since wehave not observed potent in vivo toxin neutralization by single mAbs, westudied the impact of toxin sequence variation on the potency of mAbcombinations. As with the binding studies, only mAb combinations bindingtightly to both A1 and A2 subtypes potently neutralized toxin in vivo.Thus the impact of subtype variability on potency must be evaluated inthe development of antibody based toxin therapy, whether such therapy isoligoclonal or polyclonal. Similarly, toxin vaccines based on a singlesubtype may need to be evaluated for their ability to protect againstrelated subtypes.

An unexpected finding in these studies was that mAbs binding to thetranslocation domain and/or catalytic domains of BoNT had neutralizingactivity, either when combined with each other or when combined with amAb recognizing the BoNT receptor binding domain (HC). Neutralizingactivity has also been reported for mAbs binding the catalytic domain oftetanus toxin (Kozaki et al. (1995) Microbiol. Immunol., 39: 767-774)and ricin (Lang et al. (1993) J. Immunol. 151: 466-472). Since mAbswhich do not bind to the BoNT receptor binding domain cannot strictlyblock the interaction of BoNT binding domain epitopes to cellularreceptors and subsequent BoNT endocytosis, the mechanism by which theycontribute to neutralization remains unknown. Possibilities includeenhancement of BoNT clearance from the circulation upon binding ofmultiple mAbs (Montero-Julian et al. (1995) Blood 85: 917-924),interference of receptor binding by a steric effect, interference withrequired intracellular toxin processes (endosomal escape or catalyticactivity) (Koriazova and Montal (2003) Nat. Struct. Biol., 10: 13-18),and/or altering intracellular BoNT trafficking. Regardless of themechanism, the ability of non-binding domain mAbs to neutralize toxinsignificantly increases the number of epitopes available forneutralizing mAb generation, increasing the likelihood of finding mAbsbinding and neutralizing all BoNT subtypes.

While we only studied the impact of sequence variability on antibodybinding and neutralization for a single serotype (BoNT/A), threeserotypes (BoNT/C, D, and F) have subtypes which differ from each otherby more than the 10% difference between BoNT/A1 and BoNT/A2 (10.7% to31.6%). For these three serotypes, the impact of sequence variability onimmune recognition is likely to be greater than for BoNT/A. For twoserotypes (BoNT/B and E), sequence variability was less than observedfor BoNT/A (2.6% to 7.6%). The impact of this level of sequencevariability will need to be evaluated, but is clearly in a range thatcould affect mAb binding, as shown in previous evaluations of mAbbinding to BoNT/B toxin (Gibson et al. (1988) J. Appl. Bacteriol., 64:285-291; Kozaki et al. (1998) Infect. Immun., 66: 4811-4816) and BoNT E(Kozaki et al. (1986) Infect. Immun., 52: 786-791).

In conclusion, we report the existence of considerable sequencevariability within six of the seven botulinum neurotoxin serotypes andshow that this level of variability can significantly affect antibodybinding and neutralization. Determining the full extent of such toxindiversity is an important step in the development of immunologicalbotulinum toxin assays, therapeutics and vaccines. Once the sequencevariability has been defined, it is likely that some number of thesetoxin variants will need to be produced for validation of detectionassays, therapeutics, and vaccines.

Example 6 Neutralizing Antibodies Selected and Evolved for CrossNeutralization of BoNT/A Subtypes A1, A2, and A3

The discovery of different subtypes of botulinum neurotoxins, includingBoNT/A, poses a challenge for the development of diagnostic andtherapeutic antibodies. Ideally, mAbs or mixtures of mAbs would bind toand detect/neutralize most or all of the different BoNT subtypes. Thiswould result in a detection system that did not miss the detection ofsome subtypes. For therapeutic antibodies, cross reactivity ensures thatthe antibody does not fail to neutralize one or more of the subtypes.

Selection of Antibodies Binding BoNT/A1 and BoNT/A2

To generate monoclonal antibodies capable of binding BoNT/A1 andBoNT/A2, immune phage or yeast scFv antibody libraries were sequentiallyselected, first on BoNT/A1 and then on BoNT/A2. After multiple rounds ofselection, phage or yeast antibodies were screened for binding to bothBoNT subtypes. Two scFv antibodies were identified that bound bothBoNT/A1 and BoNT/A2 with comparable affinities, (ING1, scFv KDBoNT/A1=1.17×10⁻⁹ M; scFv KD BoNT/A2=1.18×10⁻⁹ M: and ING2, scFv KDBoNT/A1=4.17×10⁻¹⁰ M; scFv KD BoNT/A2=4.5×10⁻¹⁰ M. See Table 13 forsequences of ING1 and ING2. For in vivo studies, these two scFv wereconverted to IgG. The IgG maintained high affinity binding for both A1and A2 BoNT (Table 21).

TABLE 21 Affinities of cross reactive IgG binding both BoNT/A1 and A2with high affinity. Affinities and binding kinetics were determined byflow fluorimetry. Antibody Antigen Kd On Rate Off Rate CR-1 A1 2.96 pM3.54e⁶ 1.06e⁻⁵ CR-1 A2 1.73 nM 1.62e⁷ 2.81e⁻² ING-1 A1  314 pM 2.02e⁵6.35e⁻⁵ ING-1 A2  719 pM ING-2 A1 9.57 pM 1.09e⁶ 1.05e⁻⁵ ING-2 A2 7.42pM 9.78e⁵ 7.26e⁻⁶Generation of a HuC25 Variant Capable of Binding Both BoNT/A1 and A2With High Affinity.

Neither HuC25 nor its higher affinity derivatives bind BoNT/A2 with highaffinity (see Table 22 for affinities of AR2 for BoNT/A1 and BoNT/A2).To increase affinity for BoNT/A2, we started with the higher affinityvariant AR2. This antibody as an IgG has a more than 10,000 loweraffinity for BoNT/A2 than BoNT/A1 and a very low affinity for BoNT/A2 of2.0×10⁻⁷ M (Table 22).

TABLE 22 Affinity and binding kinetics of AR2 IgG and yeast displayedscFv for BoNT/A1 and BoNT/A2. BoNT/A1 BoNT/A2 Method K_(d) (M⁻¹) k_(on)(M⁻¹ s⁻¹) k_(off) (s⁻¹)c K_(d) (M⁻¹) k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹)IgG/SPR in 1.46 × 10⁻¹¹  1.09 × 10⁶  1.6 × 10⁻⁵  1.7 × 10⁻⁷ 2.0 × 10⁴3.4 × 10⁻³ BIAcore IgG/Kinexa 6.8 × 10⁻¹² 3.69 × 10⁶ 2.66 × 10⁻⁵ 2.01 ×10⁻⁷ ScFv yeast 6.1 × 10⁻¹¹ 1.08 × 10⁻⁷ display

Libraries of AR2 mutants were generated using spiked oligonucleotides orerror prone PCR and the mutants displayed on the surface of yeast asscFv. Libraries were serially selected, first on BoNT/A1 and BoNT/A2,yielding the mutants WR1(T), K_(D) BoNT/A2=3.7 nM, WR1(V), K_(D)BoNT/A2=9.0 nM and CR1 K_(D) BoNT/A2=850 pM (Table 20 for sequences).The highest affinity scFv, CR1 was converted to an IgG, which had anapproximately 80 fold higher affinity for BoNT/A2 toxin compared to theparental AR2 IgG, while also increasing its affinity for BoNT/A1approximately 10 fold (Table 23). To further increase affinity forBoNT/A2, the CR1 scFv gene was randomly mutated, displayed on yeast andhigher affinity scFv selected sequentially on BoNT/A1 and BoNT/A2,yielding the mutant CR2 (see sequence listing or Table 13 for sequence).After conversion to an IgG, CR2 had an approximately 6 fold higheraffinity for BoNT/A2 than CR1 and maintained high affinity binding forBoNT/A1 (Table 23).

TABLE 23 Affinities and binding kinetics of AR2, CR1, and CR2 IgG forBoNT/A1 and BoNT/A2 as determined by flow flourimetry, BoNT/A1 BoNT/A2Method K_(d) (M⁻¹) k_(on) (M⁻¹ s⁻¹) k_(off) (s⁻¹) K_(d) (M⁻¹) k_(on)(M⁻¹ s⁻¹) k_(off) (s⁻¹) AR2  6.8 × 10⁻¹² 3.69 × 10⁶ 2.66 × 10⁻⁵ 2.01 ×10⁻⁷ CR1 2.96 × 10⁻¹² 3.54 × 10⁶ 1.06 × 10⁻⁵ 1.73 × 10⁻⁹ 1.62 × 10⁷ 2.81× 10⁻² CR2  2.9 × 10⁻¹⁰Antibodies With Higher Affinity for BoNT/A2 Neutralize BoNT/A2 With HighPotency.

In example 5, it was shown that 50 ug of the combination of antibodiesHuC25, B4, and 3D12 could neutralize 40,000 mouse LD50s of BoNT/A1 butless than 200 LD50s of BoNT/A2. Neither B4 nor HuC25 bound BoNT/A2 withhigh affinity. We therefore studied the ability of the CR1 antibody,derived from HuC25 but with high affinity for BoNT/A1 and BoNT/A2,combined with RAZ1, and either ING1 or ING2 to neutralize BoNT/A1 andBoNT/A2. Using an antibody dose of 50 ug total antibody, the combinationof CR1+RAZ1+either ING1 or ING2 completely protected mice challengedwith 40,000 mouse LD50s of BoNT/A1. The same doses of antibody showedsignificant protection of mice challenged with BoNT/A2, with thecombination CR1+RAZ1+ING1 being the most potent, completely protectingmice challenged with 40,000 mouse LD50s of BoNT/A2 (FIG. 25). Thus wehave shown that it is possible to generate as well as evolve antibodiesthat can bind multiple BoNT subtypes with high affinity, in this caseBoNT/A1 and A2, and that this leads to potent neutralization when theantibodies are combined

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of neutralizing Botulinum neurotoxin (BoNT) in a mammal saidmethod comprising administering to said mammal at least two differentantibodies for a BoNT serotype, wherein a first antibody of said twodifferent antibodies comprises: a) a V_(H)CDR1 comprising the amino acidsequence of SEQ ID NO:49; b) a V_(H)CDR2 comprising the amino acidsequence of SEQ ID NO:51; c) a V_(H)CDR3 comprising the amino acidsequence of SEQ ID NO:75; d) a V_(L)CDR1 comprising the amino acidsequence of SEQ ID NO:105, or SEQ ID NO:109; e) a V_(L)CDR2 comprisingthe amino acid sequence of SEQ ID NO:99, SEQ ID NO:107, or SEQ IDNO:111; and f) a V_(L)CDR3 comprising the amino acid sequence of SEQ IDNO:131 or SEQ ID NO:134.
 2. The method of claim 1, wherein said BoNTserotype is BoNT/A.
 3. The method of claim 1, wherein said firstantibody binds at least two different subtypes selected from the groupconsisting of BoNT/A1, BoNT/A2, and BoNT/A3 each with an affinitygreater than about 10 nM.
 4. The method of claim 1, wherein said firstantibody binds BoNT/A1 and BoNT/A2 each with an affinity greater thanabout 10 nM.
 5. The method of claim 4, wherein the at least twodifferent antibodies simultaneously bind at least one of subtypesBoNT/A1 and BoNT/A2.
 6. The method of claim 1, wherein said firstantibody comprises: a) a V_(L)CDR1 comprising the amino acid sequence ofSEQ ID NO:105; b) a V_(L)CDR2 comprising the amino acid sequence of SEQID NO:99; and c) a V_(L)CDR3 comprising the amino acid sequence of SEQID NO:131.
 7. The method of claim 6, wherein said first antibodycomprises: a) a heavy chain comprising the amino acid sequence of SEQ IDNO:12; and b) a light chain comprising the amino acid sequence of SEQ IDNO:13.
 8. The method of claim 1, wherein said first antibody comprises:a) a V_(L)CDR1 comprising the amino acid sequence of SEQ ID NO:105; b) aV_(L)CDR2 comprising the amino acid sequence of SEQ ID NO:107; and c) aV_(L)CDR3 comprising the amino acid sequence of SEQ ID NO:131.
 9. Themethod of claim 8, wherein said first antibody comprises: a) a heavychain comprising the amino acid sequence of SEQ ID NO:12; and b) a lightchain comprising the amino acid sequence of SEQ ID NO:14.
 10. The methodof claim 1, wherein said first antibody comprises: a) a V_(L)CDR1comprising the amino acid sequence of SEQ ID NO:109; b) a V_(L)CDR2comprising the amino acid sequence of SEQ ID NO:111; and c) a V_(L)CDR3comprising the amino acid sequence of SEQ ID NO:134.
 11. The method ofclaim 10, wherein said first antibody comprises: a) a heavy chaincomprising the amino acid sequence of SEQ ID NO:12; and b) a light chaincomprising the amino acid sequence of SEQ ID NO:15.
 12. The method ofclaim 1, wherein at least one of said two different antibodies is asingle chain Fv (scFv).
 13. The method of claim 1, wherein at least oneof said two different antibodies is an IgG.
 14. The method of claim 1,wherein at least one of said two different antibodies is a Fab.
 15. Themethod of claim 1, wherein at least one of said two different antibodiesis a (Fab′)₂.
 16. The method of claim 1, wherein at least one of saidtwo different antibodies is a (scFv′)₂.
 17. The method of claim 1,wherein a second antibody of said two different antibodies is selectedfrom the group consisting of: a) an antibody comprising i) a heavy chaincomprising the amino acid sequence of SEQ ID NO:11 and ii) a light chaincomprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:354; b)an antibody comprising i) a heavy chain comprising the amino acidsequence of SEQ ID NO:9 or SEQ ID NO:23 and ii) a light chain comprisingthe amino acid sequence of SEQ ID NO:22; c) an antibody comprising i) aheavy chain comprising the amino acid sequence of SEQ ID NO:12, and ii)a light chain comprising the amino acid sequence of SEQ ID NO:358, SEQID NO:13, SEQ ID NO:14, or SEQ ID NO:15; or d) an antibody comprising i)a heavy chain comprising the amino acid sequence of SEQ ID NO:16 and ii)a light chain comprising the amino acid sequence of SEQ ID NO:362. 18.The method of claim 1, wherein a second antibody of said two differentantibodies is selected from the group consisting of: a) an antibodycomprising i) a heavy chain comprising the amino acid sequence of SEQ IDNO:11 and ii) a light chain comprising the amino acid sequence of SEQ IDNO: 10 or SEQ ID NO:354; b) an antibody comprising i) a heavy chaincomprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:23 andii) a light chain comprising the amino acid sequence of SEQ ID N022 orc) an antibody comprising i) a heavy chain comprising the amino acidsequence of SEQ ID NO:16 and ii) a light chain comprising the amino acidsequence of SEQ ID NO:362.
 19. A composition for neutralizing aBotulinum neurotoxin (BoNT), said composition comprising at least twodifferent antibodies for a BoNT serotype, wherein a first antibody ofsaid two different antibodies comprises: a) a V_(H)CDR1 comprising theamino acid sequence of SEQ ID NO:49; b) a V_(H)CDR2 comprising the aminoacid sequence of SEQ ID NO:51; c) a V_(H)CDR3 comprising the amino acidsequence of SEQ ID NO:75; d) a V_(L)CDR1 comprising the amino acidsequence of SEQ ID NO:105 or SEQ ID NO:109; e) a V_(L)CDR2 comprisingthe amino acid sequence of SEQ ID NO:99, SEQ ID NO:107, or SEQ IDNO:111; and f) a V_(L)CDR3 comprising the amino acid sequence of SEQ IDNO:131 or SEQ ID NO:134.
 20. The composition of claim 19, wherein saidBoNT serotype is BoNT/A.
 21. The composition of claim 19, wherein saidfirst antibody binds at least two different subtypes selected from thegroup consisting of BoNT/A1, BoNT/A2, and BoNT/A3 each with an affinitygreater than about 10 nM.
 22. The composition of claim 19, wherein saidfirst antibody binds BoNT/A1 and BoNT/A2 each with an affinity greaterthan about 10 nM.
 23. The composition of claim 19, wherein the at leasttwo different antibodies simultaneously bind at least one of subtypesBoNT/A1 and BoNT/A2.
 24. The composition of claim 19, wherein said firstantibody comprises: a) a V_(L)CDR1 comprising the amino acid sequence ofSEQ ID NO:105; b) a V_(L)CDR2 comprising the amino acid sequence of SEQID NO:107; and c) a V_(L)CDR3 comprising the amino acid sequence of SEQID NO:131.
 25. The composition of claim 24, wherein said first antibodycomprises: a) a heavy chain comprising the amino acid sequence of SEQ IDNO:12; and b) a light chain comprising the amino acid sequence of SEQ IDNO:14.
 26. The composition of claim 19, wherein at least one of said twodifferent antibodies is a single chain Fv (scFv).
 27. The composition ofclaim 19, wherein at least one of said two different antibodies is anIgG.
 28. The composition of claim 19, wherein at least one of said twodifferent antibodies is a Fab.
 29. The composition of claim 19, whereinat least one of said two different antibodies is a (Fab′)₂.
 30. Thecomposition of claim 19, wherein at least one of said two differentantibodies is a (scFv′)₂.
 31. The composition of claim 19, wherein asecond antibody of said two different antibodies is selected from thegroup consisting of: a) an antibody comprising i) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 11 and ii) a lightchain comprising the amino acid sequence of SEQ ID NO: 10 or SEQ IDNO:354; b) an antibody comprising i) a heavy chain comprising the aminoacid sequence of SEQ ID NO:9 or SEQ ID NO:23 and ii) a light chaincomprising the amino acid sequence of SEQ ID NO:22; c) an antibodycomprising i) a heavy chain comprising the amino acid sequence of SEQ IDNO:12 and ii) a light chain comprising the amino acid sequence of SEQ IDNO:358, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15; or d) an antibodycomprising i) a heavy chain comprising the amino acid sequence of SEQ IDNO: 16 and ii) a light chain comprising the amino acid sequence of SEQID NO:362.
 32. The composition of claim 19, wherein a second antibody ofsaid two different antibodies is selected from the group consisting of:a) an antibody comprising i) a heavy chain comprising the amino acidsequence of SEQ ID NO:11 and ii) a light chain comprising the amino acidsequence of SEQ ID NO:10 or SEQ ID NO:354; b) an antibody comprising i)a heavy chain comprising the amino acid sequence of SEQ ID NO:9 or SEQID NO:23 and ii) a light chain comprising the amino acid sequence of SEQID NO:22; or c) an antibody comprising i) a heavy chain comprising theamino acid sequence of SEQ ID NO: 16 and ii) a light chain comprisingthe amino acid sequence of SEQ ID NO:362.
 33. The composition of claim19, wherein said antibodies are in a pharmaceutically acceptableexcipient.
 34. The composition of claim 33, wherein said composition isa unit dosage formulation.
 35. An isolated antibody that binds Botulinumneurotoxin (BoNT), wherein said antibody comprises: a) a V_(H)CDR1comprising the amino acid sequence of SEQ ID NO:49; b) a V_(H)CDR2comprising the amino acid sequence of SEQ ID NO:51; c) a V_(H)CDR3comprising the amino acid sequence of SEQ ID NO:75; d) a V_(L)CDR1comprising the amino acid sequence of SEQ ID NO:105 or SEQ ID NO:109; e)a V_(L)CDR2 comprising the amino acid sequence of SEQ ID NO:99, SEQ IDNO:107, or SEQ ID NO:111; and f) a V_(L)CDR3 comprising the amino acidsequence of SEQ ID NO:131 or SEQ ID NO:134.
 36. The antibody of claim35, wherein said antibody binds both the BoNT/A1 and BONT/A2 subtypeseach with an affinity greater than about 10 nM.
 37. The isolatedantibody of claim 35, wherein said antibody comprises: a) a V_(L)CDR1comprising the amino acid sequence of SEQ ID NO:105; b) a V_(L)CDR2comprising the amino acid sequence of SEQ ID NO:107; and c) a V_(L)CDR3comprising the amino acid sequence of SEQ ID NO:131.
 38. The isolatedantibody of claim 37, wherein said antibody comprises: a) a heavy chaincomprising the amino acid sequence of SEQ ID NO:12; and b) a light chaincomprising the amino acid sequence of SEQ ID NO:14.
 39. The antibody ofclaim 35, wherein said antibody is a single chain Fv (scFv).
 40. Theantibody of claim 35, wherein said antibody is an IgG.
 41. The antibodyof claim 35, wherein said antibody is a Fab.
 42. The antibody of claim35, wherein said antibody is a (Fab′)₂.
 43. The antibody of claim 35,wherein said antibody is a (scFv′)₂.
 44. A kit for neutralizing aBotulinum neurotoxin, said kit comprising: a composition according toany of claims 19, 20-25 and 26-34; and instructional materials teachingthe use of said composition to neutralize a Botulinum neurotoxin. 45.The kit of claim 44, wherein said composition is stored in a disposablesyringe.
 46. The composition of claim 19, wherein said first antibodycomprises: a) a V_(L)CDR1 comprising the amino acid sequence of SEQ IDNO:105; b) a V_(L)CDR2 comprising the amino acid sequence of SEQ IDNO:99; and c) a V_(L)CDR3 comprising the amino acid sequence of SEQ IDNO:131.
 47. The composition of claim 46, wherein said first antibodycomprises: a) a heavy chain comprising the amino acid sequence of SEQ IDNO:12; and b) a light chain comprising the amino acid sequence of SEQ IDNO:13.
 48. The composition of claim 19, wherein said first antibodycomprises: a) a V_(L)CDR1 comprising the amino acid sequence of SEQ IDNO:109; b) a V_(L)CDR2 comprising the amino acid sequence of SEQ IDNO:111; and c) a V_(L)CDR3 comprising the amino acid sequence of SEQ IDNO:134.
 49. The composition of claim 48, wherein said first antibodycomprises: a) a heavy chain comprising the amino acid sequence of SEQ IDNO:12; and b) a light chain comprising the amino acid sequence of SEQ IDNO:15.
 50. The isolated antibody of claim 35 comprising: a) a V_(L)CDR1comprising the amino acid sequence of SEQ ID NO:105; b) a V_(L)CDR2comprising the amino acid sequence of SEQ ID NO:99; and c) a V_(L)CDR3comprising the amino acid sequence of SEQ ID NO:131.
 51. The isolatedantibody of claim 50 comprising: a) a heavy chain comprising the aminoacid sequence of SEQ ID NO:12; and b) a light chain comprising the aminoacid sequence of SEQ ID NO:13.
 52. The isolated antibody of claim 35comprising: a) a V_(L)CDR1 comprising the amino acid sequence of SEQ IDNO:109; b) a V_(L)CDR2 comprising the amino acid sequence of SEQ IDNO:111; and c) a V_(L)CDR3 comprising the amino acid sequence of SEQ IDNO:134.
 53. The isolated antibody of claim 52 comprising: a) a heavychain comprising the amino acid sequence of SEQ ID NO:12; and b) a lightchain comprising the amino acid sequence of SEQ ID NO:15.